Experiences with infectious cDNA clones of equine arteritis virus: lessons learned and insights gained

Abstract

The advent of recombinant DNA technology, development of infectious cDNA clones of RNA viruses, and reverse genetic technologies have revolutionized how viruses are studied. Genetic manipulation of full-length cDNA clones has become an especially important and widely used tool to study the biology, pathogenesis, and virulence determinants of both positive and negative stranded RNA viruses. The first full-length infectious cDNA clone of equine arteritis virus (EAV) was developed in 1996 and was also the first full-length infectious cDNA clone constructed from a member of the order Nidovirales. This clone was extensively used to characterize the molecular biology of EAV and other Nidoviruses. The objective of this review is to summarize the characterization of the virulence (or attenuation) phenotype of the recombinant viruses derived from several infectious cDNA clones of EAV in horses, as well as their application for characterization of the molecular basis of viral neutralization, persistence, and cellular tropism.

Keywords: Arteriviruses; EAV; EVA; Equine arteritis virus; Equine viral arteritis; Reverse genetics; cDNA clones.

Fig. 1

Passage history of the parental virulent Bucyrus strain of EAV. (H = Passaged in horse; HK = Primary horse kidney cell, RK = Primary rabbit kidney cell passage, ED = Equine dermis cell [NBL-6; ATCC CCL-57]).

Fig. 2

Most commonly used infectious cDNA clones of EAV.

Fig. 3

EAV genome organization (a) and virion architecture (b). (a) The genomic open reading frames (ORFs) are indicated and the names of the corresponding proteins are depicted. The pink boxes represent the body transcription regulatory sequences (TRSs). The papain-like cysteine protease (PCPβ), papain-like protease domain 2 (PLP2 [previously known as cysteine protease; CP] that is predicted to contain the ovarian tumor domain-containing [OTU] superfamily of deubiquitinating enzymes [DUBs] on the basis of comparative sequence analysis) and serine protease (SP) are located in the nsp1, nsp2 and nsp4 of viral replicase, respectively. The nested set of mRNAs that is found in infected cells is depicted below the genome, with RNA1 being identical to the viral genome and sgmRNAs 2 to 7 being used to express the structural protein genes located in the 3′-proximal quarter of the genome. The light blue box at the 5′ end of each sgmRNA represents the common leader sequence, which is derived from the 5′ end of the genome. With the exception of the bicistronic sgmRNAs 2 and 5, the sgmRNAs are functionally monocistronic. Translation of proteins from sgmRNAs 2 (E and GP2 proteins) and 5 (ORF5a protein and GP5) by leaky scanning of the 5′-proximal end of these sgmRNAs (Firth et al., 2011, Snijder et al., 1999). The ORFs 1a and 1b located at the 5′ end of the genome are translated into two polyproteins (pp1a and pp1ab) that are further processed into 12–13 nonstructural proteins by three viral proteases (nsps 1, 2, and 4). (b) EAV particle consists of a nucleocapsid (N) and seven envelope proteins which include two major envelope proteins (GP5 and M form a dimer), three minor envelope glycoproteins (GP2, GP3, and GP4 form a trimer), and two other minor envelope proteins (E and ORF5a protein). Adapted from Balasuriya et al. (2013) with permission.

Fig. 4

Comparative amino acid sequence analysis between the EAV VBS and attenuated EAV strains derived from it identified several amino acid substitutions in both non-structural (A) and structural proteins (B and C). The putative glycosylation sites in GP2 (Asn-155), GP3 (Asn-28, Asn-29, Asn-49, Asn-96, Asn-106, and Asn-118), GP4 (Asn-33, Asn-55, Asn-65, and Asn-90), and GP5 (Asn-56, Asn-71, and Asn-81) are depicted in blue dots. The most significant amino acid changes are highlighted (blue and bold) in the figure.

Fig. 5

Ability to establish persistent infection in HeLa-L cell line with recombinant viruses rVBS/P80NS4m, rVBS/P80S, rVBS/P80ORFs2ab, rVBS/P80ORFs34, rVBS/P80ORFs234, and rVBS/GP5P98→L. Tissue culture supernatants from serial subculture up to the 10th passage were harvested and titrated. The representative data of two separate experiments are shown. Adapted from Zhang et al., 2008a, Zhang et al., 2008c with permission.

Fig. 6

Infection of lymphocytes and monocytes with chimeric EAV viruses. The genome of the infectious full-length cDNA clone of rVBS (red boxes) and the genome of the rMLV clone (blue boxes) are depicted. The genes encoding structural proteins of EAV HK116 virus are shown in green. The four chimeric viruses containing nonstructural and structural protein genes of either rVBS, or rMLV virus are also depicted. L, leader; An, poly A tail. The CD3⁺ T lymphocytes and CD14⁺ monocytes infected with recombinant viruses rVBS (panels a and b), rVBS/HK116 S (panels c and d), rMLV (panels e and f), rVBS/MLV S (panels g and h), rMLV/VBS S (panels i and j), rMLV/VBS 234 (panels k and l) and rMLV/VBS 56 (panels m and n) were examined by dual-color immunofluorescence flow cytometric analysis using MAbs against EAV nsp1 (12A4) and MAbs for cell specific cell surface antigens at 24 hours post infection (hpi). Adapted from Go et al. (2010) with permission.