Zhengchun Lu

Formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes.

ABSTRACT The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by cleavage of the nsp2/3 junction by the nsp2 protease, was nonessential for the formation of DMVs. However, the substitution of each of a cluster of four conserved cysteine residues, residing in a predicted luminal loop of nsp3, completely blocked DMV formation. Some of these mutant nsp3 proteins were also found to be highly cytotoxic, in particular, exerting a dramatic effect on the endoplasmic reticulum. The functionality of an engineered N glycosylation site in the cysteine-containing loop confirmed both its presence in the lumen and the transmembrane nature of nsp3. This mutant displayed an interesting intermediate phenotype in terms of DMV formation, with paired and curved membranes being formed, but DMV formation apparently being impaired. The effect of nsp3 mutations on replicase polyprotein processing was investigated, and several mutations were found to influence processing of the region downstream of nsp3 by the nsp4 main protease. When tested in an EAV reverse genetics system, none of the nsp3 mutations was tolerated, again underlining the crucial role of the protein in the arterivirus life cycle.

Molecular epidemiology and genetic characterization of equine arteritis virus isolates associated with the 2006-2007 multi-state disease occurrence in the USA

In 2006-2007, equine viral arteritis (EVA) was confirmed for the first time in Quarter Horses in multiple states in the USA. The entire genome of an equine arteritis virus (EAV) isolate from the index premises in New Mexico was 12 731 nt in length and possessed a previously unrecorded unique 15 nt insertion in the nsp2-coding region in ORF1a and a 12 nt insertion in ORF3. Sequence analysis of additional isolates made during this disease occurrence revealed that all isolates from New Mexico, Utah, Kansas, Oklahoma and Idaho had 98.6-100.0 % (nsp2) and 97.8-100 % (ORF3) nucleotide identity and contained the unique insertions in nsp2 and ORF3, indicating that the EVA outbreaks in these states probably originated from the same strain of EAV. Sequence and phylogenetic analysis of several EAV isolates made following an EVA outbreak on another Quarter Horse farm in New Mexico in 2005 provided evidence that this outbreak may well have been the source of virus for the 2006-2007 occurrence of the disease. A virus isolate from an aborted fetus in Utah was shown to have a distinct neutralization phenotype compared with other isolates associated with the 2006-2007 EVA occurrence. Full-length genomic sequence analysis of 18 sequential isolates of EAV made from eight carrier stallions established that the virus evolved genetically during persistent infection, and the rate of genetic change varied between individual animals and the period of virus shedding.

Development and Evaluation of One-Step TaqMan Real-Time Reverse Transcription-PCR Assays Targeting Nucleoprotein, Matrix, and Hemagglutinin Genes of Equine Influenza Virus

ABSTRACT The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of ≥10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid

Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was less sensitive (93.4%) than the World Organization for Animal Health (OIE)-prescribed VI test (gold standard). The sensitivity of both PCR assays was high (100.0% [T1] and 95.2% [T2]) for detecting EAV RNA in TCF. In light of the discrepancy in sensitivity between either real-time RT-PCR assay and VI, semen that is negative for EAV nucleic acid by real-time RT-PCR that is from an EAV-seropositive stallion should be confirmed free of virus by VI. Similarly, the presence of EAV in TCF samples that are VI-positive but real-time RT-PCR-negative should be confirmed in a 1-way neutralization test using anti-EAV equine serum or by fluorescent antibody test using monoclonal antibodies to EAV. If the viral isolate is not identified as EAV, such samples should be tested for other equine viral pathogens. The results of this study underscore the importance of comparative evaluation and validation of real-time RT-PCR assays prior to their recommended use in a diagnostic setting for the detection and identification of specific infectious agents.

New Real-Time PCR Assay Using Allelic Discrimination for Detection and Differentiation of Equine Herpesvirus-1 Strains with A2254 and G2254 Polymorphisms

ABSTRACT A single-nucleotide polymorphism (A2254 or G2254) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E2) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69–72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E1) was developed by redesigning primers and probes specific to ORF30. The E1 and E2 rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A2254 or G2254) in all archived isolates plus 168 of the clinical samples. The E1 assay was 10 times more sensitive than E2, with a lower detection limit of 10 infectious virus particles. Furthermore, all A2254 and G2254 genotypes along with samples from three cases of dual infection (A2254+G2254) were correctly identified by E1, whereas E2 produced 20 false dual positive results with only one actual mixed A2254+G2254 genotype confirmed. Based on these findings, E1 offers greater sensitivity and accuracy for the detection and A/G2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they are highly species specific and differ significantly in cellular tropism in cultured cells. In this study we examined the role of the two major envelope proteins (GP5 and M) of EAV and PRRSV in determining their cellular tropism. We generated three viable EAV/PRRSV chimeric viruses by swapping the N-terminal ectodomains of these two proteins from PRRSV IA1107 strain into an infectious cDNA clone of EAV (rMLVB4/5 GP5ecto, rMLVB4/5/6 Mecto and rMLVB4/5/6 GP5&Mecto). The three chimeric viruses could only infect EAV susceptible cell lines but not PRRSV susceptible cells in culture. Therefore, these data unequivocally demonstrate that the ectodomains of GP5 and M are not the major determinants of cellular tropism, further supporting the recent findings that the minor envelope proteins are the critical proteins in mediating cellular tropism (Tian et al., 2012).

Development and Characterization of an Infectious cDNA Clone of the Modified Live Virus Vaccine Strain of Equine Arteritis Virus

ABSTRACT A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay. In vitro studies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells. In vivo studies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals.

Evaluation of Two Magnetic-Bead-Based Viral Nucleic Acid Purification Kits and Three Real-Time Reverse Transcription-PCR Reagent Systems in Two TaqMan Assays for Equine Arteritis Virus Detection in Semen

ABSTRACT This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.