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Research in Veterinary Science 85 (2008) 359–367

Molecular characterization of the hemagglutinin-neuraminidase geneof porcine rubulavirus isolates associated with neurological disorders

in fattening and adult pigs

J.I. Sanchez-Betancourt a, G. Santos-Lopez b, R. Alonso c, J.M. Doporto a,H. Ramırez-Mendoza d, S. Mendoza e, J. Hernandez f, J. Reyes-Leyva b,*, M.E. Trujillo a,*

a Departamento de Produccion Animal ‘‘Cerdos’’, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autonoma de Mexico (UNAM),

Av. Universidad No. 3000, Col. Copilco, Coyoacan, 04510 Mexico, D.F., Mexicob Laboratorio de Virologıa, Centro de Investigacion Biomedica de Oriente, Instituto Mexicano del Seguro Social,

Km 4.5 Carretera Atlixco-Metepec, CP 74360 Metepec, Puebla, Mexicoc Departamento de Genetica Molecular, Facultad de Medicina Veterinaria y Zootecnia, UNAM, Mexico, D.F., Mexico

d Departamento de Microbiologıa e Inmunologıa, Facultad de Medicina Veterinaria y Zootecnia, UNAM, Mexico, D.F., Mexicoe Coordinacion de Estudios de Posgrado, Facultad de Estudios Superiores Cuautitlan, UNAM, Mexico, D.F., Mexico

f Laboratorio de Inmunologıa, Centro de Investigacion en Alimentacion y Desarrollo, A.C., Hermosillo, Sonora, Mexico

Accepted 11 October 2007

Abstract

‘‘Blue eye disease’’ is a viral infection of swine endemic in Mexico, which produces fatal encephalitis accompanied by respiratory signsand corneal opacity in suckling piglets. An atypical blue eye disease outbreak presented high rates of neurological signs in fattening andadult pigs from 2000 to 2003. In order to identify the basis of increased neurovirulence, the hemagglutinin-neuraminidase (HN) gene ofseveral porcine rubulavirus isolates were sequenced and compared with that of La Piedad Michoacan virus and other isolates that did notproduce neurological disorders in weaned pigs. Nine amino acid mutations distinguished the high neurovirulent PAC6–PAC9 viruses,whereas five mutations characterized the low neurovirulent PAC2 and PAC3 viruses. HN protein three-dimensional models showed thatthe main conformation and functional domains were preserved, although substitutions A223T and A291D occurred in PAC2 and PAC3viruses, as well as A511K and E514K presented in PAC6–PAC9 viruses considerably modified the properties of the HN protein surface.The increased positive charge of the HN protein of PAC6–PAC9 viruses seems to be associated with their increased neurovirulence.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Porcine rubulaviruses; Hemagglutinin-neuraminidase; Sequencing; Protein structure; Structure–function analysis

1. Introduction

Blue eye disease (BED) is a viral infection of swine thatemerged in La Piedad Michoacan Mexico in 1980. The cau-sal agent of BED was recognized as a paramyxovirus andcalled La Piedad Michoacan virus (LPMV) (Moreno-Lopez et al., 1986; Stephano et al., 1988). Gene sequencing

0034-5288/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.rvsc.2007.10.007

* Corresponding authors. Tel./fax: +52 55 56 225 951.E-mail addresses: jreyesleyva@correo.unam.mx (J. Reyes-Leyva),

elenam@servidor.unam.mx (M.E. Trujillo).

data showed that LPMV was closely related to Mumpsvirus (MuV), Simian virus 5 (SV5), Parainfluenza virustype 2 (PIV-2) and type 4 (PIV-4). This led to the classifi-cation of LPMV as a porcine species in the Rubulavirusgenus, Paramyxoviridae family (Sundqvist et al., 1992;Linne et al., 1992; Rima et al., 1995).

The first BED outbreaks were characterized by a neuro-logical and respiratory syndrome with corneal opacity thatmainly affected newborn and suckling pigs; morbidity andmortality rates reached 20% and 90%, respectively.Abortions and mummified fetuses were also found in some

360 J.I. Sanchez-Betancourt et al. / Research in Veterinary Science 85 (2008) 359–367

pregnant sows, but signs of central nervous system (CNS)involvement were not found in weaned, fattening or maturepigs (Stephano et al., 1988).

Experimental infection with LPMV provoked an acutefatal encephalomyelitis with interstitial pneumonia andcorneal opacity in newborn pigs. Neither LPMV nor anyof the first porcine rubulavirus (PoRV) isolates producedclinical signs if they were inoculated in pigs of more than1 month of age. In addition, the spread of LPMV intothe CNS was broader if pigs were inoculated at 3-day-oldthan at 17-day-old (Moreno-Lopez et al., 1986; Stephanoet al., 1988; Hernandez-Jauregui et al., 1992; Allan et al.,1996).

PoRV strain PAC3 was isolated in 1992 during a BEDoutbreak that presented high rates of female and maleinfertility. Experimental inoculation with PAC3 virus pro-duced epididymis swelling, sperm granuloma and testisatrophy in adult boars and spreading via the sperm frac-tion of infected semen. In gilts inoculated during pregnancycaused fetal death and mummification. However, PAC3virus did not produce neurological signs in either weanedor adult pigs infected experimentally and showed low neu-rovirulence in suckling pigs (Ramırez-Mendoza et al., 1997;Reyes-Leyva et al., 2002; Hernandez-Jauregui et al., 2004;Solıs et al., 2007).

Signs of CNS involvement were rarely observed inweaned pigs during the BED outbreaks that occurredbefore 1990 although their frequency has risen during thelast decade. Changes in clinical behavior and increased vir-ulence of PoRV have been well documented (Stephano,2002). All these data indicate that PoRV are evolvingand presenting a better adaptation to mature porcinetissues.

PoRV is an enveloped virus constituted by a single-stranded negative sense RNA genome of 15 Kb, which isdivided into 6 genes that code for the structural proteins;nucleoprotein (NP), phosphoprotein (P), matrix (M),fusion (F), hemagglutinin-neuraminidase (HN) and large(L) proteins. The NP establishes a close contact with geno-mic RNA forming a helical nucleocapside. P and L pro-teins constitute the replication/transcription enzymaticcomplex, residing the RNA polymerase activity on the Lprotein while P acts as a cofactor. The M protein is local-ized in the internal side of the envelope and participates inassembling of new virions. F and HN glycoproteins areexposed in the external side of the envelope acting togetherin the penetration of the cell. Interaction of the HN glyco-protein with cell receptors conduces to virus attachmentand promoting of the F glycoprotein activity that fusesthe cell and viral membranes; this permits the ingress ofthe virus into the cell and led to formation of multinucle-ated giant cells (syncytia) observed in infected tissues(Moreno-Lopez et al., 1986; Sundqvist et al., 1990; Berget al., 1997; Linne et al., 1992; Santos-Lopez et al., 2004a).

The PoRV HN glycoprotein specifically recognizes sialyla-2,3-lactose moieties, which are located in the terminalposition of porcine nerve cell glycolipids and glycoproteins,

determining both the virus neurotropism and age-relatedswine susceptibility to infection. Its neuraminidase (siali-dase) activity contributes to virus release from infected cells(Reyes-Leyva et al., 1997, 2002; Vallejo et al., 2000; Men-doza-Magana et al., 2001; Santos-Lopez et al., 2004b).Therefore, changes in the HN glycoprotein might poten-tially affect its affinity towards porcine tissues andspreading.

Since the HN glycoprotein is the most abundant compo-nent of viral envelope it is also the main target of theimmune response induced against PoRV in infected ani-mals (Hernandez et al., 1998; Zenteno-Cuevas et al.,2007). Several works have shown that mutations affectingthe antigenic or functional domains of the HN proteinsmay be implicated in the increased neurovirulence of sev-eral paramyxoviruses (Bhaiyat et al., 1995; Palacios et al.,2005). Therefore, in the present work we analyzed andcompared the genetic sequences of several PoRVs isolatedfrom 1984–2003 in order to identify whether the observedincrease in neurovirulence can be associated with structuralchanges in the HN glycoprotein.

2. Material and methods

2.1. Viruses and cells

The following viruses were isolated at the Departmentof ‘‘Produccion Animal Cerdos’’ (PAC), Veterinary Fac-ulty, National University of Mexico: PAC2 (Jalisco/1990), PAC3 (Jalisco/1992), PAC4 (Michoacan/1993),PAC6 (Jalisco/2001), PAC7 (Jalisco/2002), PAC8 (Jali-sco/2002) and PAC9 virus (Jalisco/2003). PAC2 andPAC3 viruses were isolated during a BED outbreak thataffected breeding farms, showed low neurovirulence andproduced a notable reduction in male and female fertility(Ramırez-Mendoza et al., 1997). PAC4 virus was isolatedin a complete cycle farm showed high neurovirulence insuckling pigs with a number of neurological cases inweaned pigs. Aliquots of PAC2, PAC3 and PAC4 viruseswere preserved in liquid N2 since its isolation, they has lessthan 10 passages in cell cultures. PAC6, PAC7, PAC8 andPAC9 viruses were isolated during the outbreak occurredfrom 2000 to 2003 described below (Table 1), they havethree passages in cell cultures. Virus isolates were charac-terized by inhibition of hemagglutination, neutralizationand immunofluorescence using polyclonal antibodiesagainst porcine rubulavirus (Reyes-Leyva et al., 2002).

Vero cells were cultured in Eagle’s minimal essentialmedium supplemented with 5% fetal bovine serum. Conflu-ent cell cultures were inoculated at 1 · 105 TCID50 (tissueculture infective dose 50%) and incubated at 37 �C during72 h.

2.2. Reverse transcription and polymerase chain reaction

Infected cells were freeze-thawed three times and super-natants were clarified by centrifugation at 4500 rpm for

Table 1Neurovirulence of porcine rubulavirus isolated during several blue eye disease outbreaks

Virus name Accession number State Year Neurovirulencea References

Suckling Fattening Adults

LPMVb S77541 Michoacan 1984 20–30% – – Moreno-Lopez et al. (1986)PAC2c EF413172 Jalisco 1990 <10% – – Reyes-Leyva et al. (2002)PAC3c EF413173 Jalisco 1992 <10% – – Ramırez-Mendoza et al. (1997)PAC4d EF413174 Michoacan 1993 >30% ND – Reyes-Leyva et al. (2002)CIb AY463798 Michoacan 1991 >20% – – Paniagua-Buelnas (2000)CIIb AY487249 Michoacan 1991 >20% – – Paniagua-Buelnas (2000)CIIIb AY487251 Michoacan 1999 >20% – – Paniagua-Buelnas (2000)CIVb AY487250 Michoacan 1999 >20% – – Paniagua-Buelnas (2000)PAC6e EF413175 Jalisco 2001 10% 20% – This studyPAC7e EF413176 Jalisco 2002 10% 20% – This studyPAC8e EF413177 Jalisco 2002 10–20% 20% – This studyPAC9f EF413178 Jalisco 2003 10–15% ND 10% This study

a Indicates the percentage of neurological signs in infected pigs.b Neurological signs were just observed in suckling pigs.c These viruses produced low neurovirulence in piglets.d Neurological signs were occasionally observed in weaned pigs.e These viruses produced neurovirulence in suckling and fattening pigs.f This outbreak occurred in a farrowing farm, thus there was not available data in fattening pigs. Means neurological signs were not observed. ND data

not determined.

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15 min at 4 �C. RNA was extracted from the supernatantwith Trizol reagent following manufacturer’s instructions(Invitrogen, Carlsbad, CA).

Total RNA was retrotranscribed using Reverse Tran-scription System (Promega, Madison, WI). Briefly, themix included 1 lg of RNA, 5.8 ll of nuclease-free water,1 ll of 10 mM dNTPs and 1.25 ll of 10 lM primerORFHN-R (3 0-TTAATAGCGTGATTGAATCT-5 0, posi-tion: 1859–1840) was incubated at 65 �C/5 min. Then, 2 llof 10X first-strand buffer, 1 ll of DTT and 1 ll of RNAsinribonuclease inhibitor (40 U/ml) were added and incubatedat 42 �C/2 min. Finally, 1 ll of AMV reverse transcriptase(200 U/ ll) was added and incubated at 42 �C/50 min and70 �C/15 min. Twenty five ll of the PCR mixtures were pre-pared with 2 ll cDNA, PCR buffer (10 mM Tris–HCl pH8.3, 50 mM KCl, 3 mM 0.1% Triton X-100, and 1.5 mMMgCl2), 200 lM each dNTPs, 1 U of Taq polymerase,and 0.4 lM of each primer: ORFHN-F (5 0-CTAACAAG-GAGAACTAGGCC-3 0, position: 43–62) and ORFHN-R. The cDNA was amplified during 30 cycles in a thermalcycler (Perkin–Elmer Cetus, Gouda, The Netherlands).PCR cycle parameters were: 95 �C for 30 s, 55 �C for 30 s,and 72 �C for 2 min. An amplicon of 1817 bp was obtainedand analyzed by 1% agarose gel electrophoresis.

2.3. HN gene sequencing

PCR products were gel purified using the QIAquick gelextraction kit (Qiagen, Hilden, Germany) and sequencedin both senses with the BigDye Terminator Cycle Sequenc-ing Kit v2.0 and the ABI Prism 310 gene analyzer (AppliedBiosystems, Foster City, CA) using the following primers:HNISBF2 (5 0-ATTCGAGATCACTGTCACTG-3 0, posi-tion: 450–469), HNISBF3 (5 0-TTATTATCTTAGT-

GACGGGG-3 0, position: 803–822), HNISBF4 (5 0-CCG-TTACTTTTCAAACAGAT-3 0, position: 1208–1227),HNISBF5 (5 0-AACATGCGTTACAGGAGTAT-3 0, posi-tion: 1526–1545), HNISBR2 (5 0-ATACTCCTGTAAC-GCATGTT-3 0, position: 1526–1545), HNISBR3 (5 0-TCC-ATCTGTTTGAAAAGTAA-3 0, position: 1211–1230),HNISBR4 (5 0-CCCCGTCACTAAGATAATAA-3 0, posi-tion: 822–803), and HNISBR5 (5 0-TGGATCATTGAG-TAGCATTT-3 0, position: 502–521), and the primer pairsused for RT-PCR amplification ORFHN-F and ORFHN-R. Primer positions correspond to the LPMV HN genesequence (Sundqvist et al., 1992; accession number S77541).

Nucleotide sequences were assembled with DNASISMax 2.6 for Windows (Hitachi Software Engineering NorthAmerica, Ltd., San Francisco, CA), and uploaded in theGeneBank with the following accession numbers: PAC2(EF413172), PAC3 (EF413173), PAC4 (EF413174), PAC6(EF413175), PAC7 (EF413176), PAC8 (EF413177) andPAC9 viruses (EF413178). These sequences were comparedwith the HN gene sequence of LPMV (accession: S77541)and those of C-I, C-II, C-III and C-IV PoRV isolates(accession numbers: AY463798, AY487249, AY487251and AY487250, respectively).

2.4. Phylogenetic analysis

Gene sequences were aligned and pairwise distanceswere calculated using the DNADIST program of thePHYLIP package 3.5 (http://evolution.genetics.washing-ton.edu/phylip.html) using the Kimura two-parametermodel of nucleotide substitution. Phylogenetic trees wereconstructed using the neighbor-joining (NJ) and theUPGMA (Average Linkage clustering) methods (Felsen-stein, 1993).

Table 2Porcine rubulavirus isolation in clinically infected pigs

Pig no. Age(days)

Clinicalsigns

Brain Lung Cellpassage

Fattening pigs

1 100 R � �2 100 R � �3 100 R � + 24 115 NR + + 2,35 60 R + + 36 80 R + + 37 120 R � + 2,38 95 R + + 29 90 NR + + 1,210 105 R + + 1,211 95 NR + + 1,212 95 R � �13 100 R � �14 100 NR + + 1,215 120 R + + 316 110 R + + 3Positive

samples10/16 12/

16

Suckling pigs

17 15–20 NR + � 218 15–20 NR + � 219 15–20 R � �20 15–20 R � �21 15–20 NR + � 1,222 15–20 R � �23 15–20 NR + + 224 15–20 R + � 225 15–20 NR + + 1,226 15–20 NR + + 227 15–20 R + + 2Positive

samples8/11 4/11

Virus isolation (+) was confirmed by cytopathic effect, hemagglutinationand neutralization assays. NR, pigs presented both neurological andrespiratory signs. R, pigs presented just respiratory signs. (�), negativevirus isolation after three passage in cell cultures. (+) positive virus iso-lation at 1 first, 2 second or 3 third passage.

362 J.I. Sanchez-Betancourt et al. / Research in Veterinary Science 85 (2008) 359–367

2.5. HN protein structure prediction

Deduced amino acid sequences of PoRV HNs werealigned and compared using ClustalW 1.8 program(http://www.ebi.ac.uk/clustalw) (Chenna et al., 2003).After that, a structure-based sequence alignment was madeusing ESPript 2.2 software (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi), which shows the sequence and sec-ondary structure similarities (Gouet et al., 2003). Putative3D structures were predicted using the Swiss-Model Server(http://swissmodel.expasy.org) (Kopp and Schwede, 2004).This process was carried out by comparing the HNsequence of all PoRV isolates with the HN protein crystalof SV5 in conjunction with its neuraminidase inhibitorDANA (2,3-dehydro-2-deoxy-N-sialic acid) (PDB acces-sion: 1Z50). Molecular surface modeling of predicted HNproteins was built assuming a solvent radius of 1.4 A. Finalstructural representations were obtained with the programDeepView 3.7. The quality of predicted structures was ver-ified with the program WhatChek (Hooft et al., 1996), alsoprovided by the Swiss-Model Server. Substantially similar3D-models were obtained with Geno3D (http://geno3d-pbil.ibcp.fr/cgi-bin/geno3d_automat.pl?page) and Phyre(http://www.sbg.bio.ic.ac.uk/�phyre), but only the Swiss-Model 3D-prediction was shown.

3. Results

3.1. Description of a highly neurovirulent BED outbreak

One atypical BED outbreak occurred in the state ofJalisco, Mexico from 2000 to 2003. We analyzed a totalof 22 farms belonging to a multiple-site production sys-tem, these included 2 site 1 (breeding gestation farrowing)farms, 8 site 2 (nursery) farms and 12 site 3 (finisher)farms. The BED outbreak affected farrowing and finisherfarms. Ten to twenty percent of piglets in site 1 farms pre-sented respiratory and neurological signs. Whereas, 60%of the 3- to 4-months-old pigs in site 3 farms presentedsigns of pneumonia, 20% also presented signs of enceph-alitis (Table 1). Mortality increased 30% during theoutbreak.

A total of 27 animals were chosen for euthanasia: Elevensuckling pigs (15–20 days of age) and 16 fattening pigs(90–120 days of age). Six of the 11 suckling pigs and 4 ofthe 16 fattening pigs presented both respiratory and neuro-logical signs; all the others just presented respiratory signs.We obtained samples of both midbrain and lung tissuesfrom every euthanized animal in order to intend virusisolation. The presence of PoRV was determined by obser-vation of syncytia in infected cell cultures and hemag-glutinating activity in the supernatants; then confirmedby immunofluorescence and virus neutralization assays.PoRV isolation was positively done in 8 of 11 suckling pigsand 12 of 16 fattening pigs. All the pigs with neurologicalsigns give positive results in virus isolation (Table 2).PAC6, PAC7 and PAC8 viruses were isolated in these

farms from the lung of a fattening pig, the brain of a fatten-ing pig and the brain of a suckling pig, respectively.

Another farrowing farm belonging to a different produc-tion system was studied during the same BED outbreak.Neurological signs were observed in 10–15% of the pigletsand 10% of adult gilts. Adult mortality increased >15%during the outbreak. PAC9 virus was isolated in this farmfrom the brain of a 2-year-old gilt that presented neurolog-ical signs and corneal opacity.

3.2. HN gene sequences

HN gene sequences of PoRV isolates PAC2–PAC4 andPAC6–PAC9 were determined and compared with pub-lished sequences of LPMV and CI to CIV viruses. Varia-tion in the HN gene sequences seems to be in accordancewith the neurovirulence profile of each virus. IndeedPAC4 virus that has shown the typical signs of BED withneurovirulence in newborn and suckling pigs showed the

Fig. 1. Structure-based alignment of deduced amino acid sequences of PoRV HN proteins. Primary sequence of LPM virus is shown. Conserved residuesare dotted, and changing residues are indicated by letters. The secondary structure shown above the alignment corresponds to LPMV HN, which weretaken from the SwissModel prediction. The nomenclature biSj corresponds to the jth strand in the ith b-sheet. Helices are named according to SV5 HNcounterparts. Residues involved in neuraminidase activity are indicated by asterisks. Underlined residues are affected by the amino acid changes A291Dand E514K shown in Fig. 3. Amino acid sequence alignment was carried out with ClustalW 1.8; and the structure-based alignment with Espript 2.2.

J.I. Sanchez-Betancourt et al. / Research in Veterinary Science 85 (2008) 359–367 363

Fig. 2. Unrooted phlylogenetic tree based on the HN gene sequences ofPoRV isolates. Phylogenies were plotted with the DRAWTREE subrou-tine of the PHYLIP package. Bar, 0.002 substitutions per site.

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most conserved HN sequence with only two nucleotide (nt)changes and 99.8% identity with respect to LPMV. Allother viruses presented between 19 and 24 nt changesand 98.9–98.6% identity. PAC6–PAC9 viruses that haveshown the highest neurovirulence in fattening and adultpigs also presented the highest synonymous vs. non-synon-ymous mutation ratios compared with all other isolates(not shown). Thus the following nine amino acid (aa) sub-stitutions: R20K, V137I, A193T, V252I, N264T, V298I, G456E,A511S and E514K were exclusive for PAC6–PAC9 viruses.PAC2–PAC3 viruses that have shown the lowest neurovi-rulence in piglets presented 5 aa substitutions: A223T,A291D, S462N, S484I and I512T; two of them shared withthe CI-CIV viruses (Fig. 1).

Phylogenetic analyses of the HN gene sequences sup-ported the classification of PoRV isolates in three viral sub-groups: the first formed by LPMV and PAC4 viruses, thesecond formed by PAC2, PAC3 and CI–CIV viruses andthe third formed by PAC6–PAC9 viruses (Fig. 2). It isimportant to note that the largest amplitude shown bythe CIV branch reveals mutations induced by its previousreplication in mouse brain (Paniagua-Buelnas, 2000).

3.3. HN protein structure

The secondary structure of the PoRV HN protein waspredicted and used to perform a structure-based sequencealignment (Fig. 1). Three-dimensional (3D) models of thehead globular domain (residues 128–576) of PoRV HNproteins (Fig. 3) were constructed using as template thecrystallized SV5 HN protein (Yuan et al., 2005). The stalkregion (residues 1–127) was not modeled because its equiv-alent was not visible in the electron density maps of theSV5 HN protein. The PoRV HN protein conserved thetypical conformation of paramyxoviruses proposed byLangedijk et al. (1997) and confirmed by Crennell et al.(2000), which consists of a symmetrical b-propeller struc-ture formed by six twisted anti-parallel b-sheets organizedas a super barrel with the active site at the central region(Fig. 3a). Important residues of the active site of SV5,NDV and HuPIV-3 HN proteins were conserved in allthe PoRV isolates and corresponded to amino acids R173,R415, R503, Y531, E400–E552 and D197; therefore, the mainactivity of the HN protein seems to be preserved.

Minimal modification of the protein structure and con-formation was observed because most of the mutationswere found in protein loops or turns, except for changesat residues 221 and 223 in strand 4 of b-sheet 1 and residue370 in strand 3 of b-sheet 3; which occurred in CIV, PAC2–PAC3 and CIII viruses, respectively.

Almost half of the mutations were localized towards theC-terminal in a region comprising 16% of the protein size.It is notable that the majority of aa changes occurred onthe surface and accumulated on the external side of theprotein, on the opposite side of the oligomerization site.Some mutations considerably affected the electrostaticproperties of the HN protein surface. Indeed, substitutions

A223T and A291D presented in PAC2 and PAC3 virusesreduced the positive charge of a region that comprises res-idues I285 to W294 and I322 to G328. On the contrary, aa sub-stitution E514K found in PAC6–PAC9 viruses increased thecharacter positive of the HN protein. This mutation waslocalized at a small variable region constituted by residues511–514. Indeed, LPMV, PAC4 and CI–CIV isolates pre-sented the sequence AIGE; PAC2 and PAC3 viruses pos-sessed ATGE, whereas PAC6, PAC7, PAC8 and PAC9isolates presented SIGK (Fig. 1). The change fromAIGE511–514 to SIGK presented in PAC6–PAC9 viruseschanged the electrostatic potential of this part of the pro-tein producing a large corridor of positive charge thatinvolved residues Y337 to A344, P446, L447, and T480 toG489 (Fig. 3b and c).

4. Discussion

Clinical reports have indicated that virulence of porcinerubulaviruses is raising (Stephano, 2002). In order to iden-tify an association of increased neurovirulence withchanges in HN structure, we compared the geneticsequences and performed a structure–function analysis onthe HN glycoprotein of PoRV isolates that differed in neu-rovirulence. Our study included 12 viral sequences ofPoRV isolated during a period of 19 years, mutations atthe HN gene and protein levels allow the differentiation

Fig. 3. Three-dimensional structure prediction of HN protein. Upside view (a) and two external views (b, c) of HN proteins are shown. The ribbonstructures of LPMV are observed in the first column where a-helices are colored as blue, b-sheets as green, and turns or loops as gray. Sialic acid (DANA)was superimposed in the active site as a ballstick representation and colored as yellow. The other columns represent the HN molecular surface models forthe PoRV LPMV, PAC3 and PAC9 isolates, colored according to their electrostatic potential; red represents the positive, blue the negative and white theneutral character of the molecule. Arrows represent amino acid substitutions. Note the change in the electrostatic potential induced by A223T, A291D andE514K mutations. Structure representations were done with DeepView 3.7.

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between low and high neurovirulent viruses (Reyes-Leyvaet al., 2002). In addition, structural analyses provided someinsights regarding the contribution of HN in neuroviru-lence because a HN protein with a highly negative surfaceseems to be related with reduced neurovirulence (PAC3virus), whereas a highly positive protein seems to be asso-ciated with increased neurovirulence (PAC9 virus). In thisrespect, PoRV isolates conserved the sequential domainNRKSCS (residues 233238) (Sundqvist et al., 1992; Law-rence et al., 2004) and the conformational active site (resi-dues R173, R415, R503, Y531, E400, E552 and D197), which areinvolved in the sialic acid-binding and neuraminidase activ-ities of paramyxoviruses (Crennell et al., 2000; Connariset al., 2002; Lawrence et al., 2004; Yuan et al., 2005). Evenso, amino acid changes A511S and E514K presented inPAC6–PAC9 viruses seem to act together increasing thepositive charge of the HN protein, not in the sialic acid-binding site but in residues that promote molecular acces-sibility to cell receptors (Crennell et al., 2000). In supportof this, a similar mutation E335K that also occurred inthe accessibility site of another viral HN glycoprotein has

been implicated in increasing both neurovirulence andneuraminidase activity of mumps virus variants (Reyes-Leyva et al., 2007).

By other side, some PoRV isolates presented sevenmutations (V252I, N264T, L450R, G456E, S484I, T500I andT526A) occurred in predicted antigenic sites for the LPMVHN protein. In fact substitutions V252I and N264T werelocalized inside a highly immunogenic porcine B-cell epi-tope, which is constituted by residues 251–267 (YVATR-SETDYYAGNSPPQ) (Zenteno-Cuevas et al., 2007).These results suggest that mutations V252I and N264T pre-sented in PAC6–PAC9 viruses might be associated withantigenic variability. This would affect the results of testsfor screening of antibodies (Nordengrahn et al., 1999) aswell as the protection efficiency of vaccines based on theHN protein (Hernandez-Jauregui et al., 1992). However,these hypotheses require experimental confirmation.

Phylogenetic analyses of HN sequences evidenced thatPoRV have evolved in three different branches: PAC6–PAC9 constitute a viral subgroup distinguished by ninecharacteristic mutations, whereas PAC2, PAC3, CI–CIV

366 J.I. Sanchez-Betancourt et al. / Research in Veterinary Science 85 (2008) 359–367

viruses constitute another subgroup. The third subgroupmay be constituted by viruses that preserve high identitywith LPMV and PAC4 viruses.

Finally, it is important to note that a lot of factors areinvolved in the clinical behavior of a viral infection not justthe genetic sequences; i.e. age, nutrition, immune response,production system, virus strain, co-infections, etc. How-ever, genetic and molecular analyses contribute to identifyviral and cellular factors involved in pathogenesis andvirulence.

5. Conclusion

This paper demonstrates a possible association betweenmutations observed in the HN gene of porcine rubulavirusstrains PAC6–PAC9 and their increased neurovirulence.

Acknowledgements

The authors acknowledge Drs. Carmen Mercado andRosalba Carreon for their technical help, and Dr. AlfredoBecerra for his support with farmers to perform the study.This work was partially financed by Proteına Animal S.Ade C.V.

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