Laura Silva-Rosales

Coat protein sequence comparisons of three Mexican isolates of papaya ringspot virus with other geographical isolates reveal a close relationship to American and Australian isolates

Summary. The coat protein gene of the papaya ringspot virus was cloned and sequenced in three Mexican isolates (two from Veracruz, and one from Chiapas). The sequences of these viral isolates were compared to those of eleven isolates from other parts of the world. They had higher similarity to isolates from Australia and the United States than to Asian isolates. A region of about one hundred nucleotides neighboring the putative aphid transmission triplet of the coat protein, contained repeats of an EK (glutamic acid-lysine) motif in all the sequences. The bearing of this region on the genetic relationships and geographical distribution of the isolates is analyzed and discussed.

Distribution of Papaya ringspot virus and Papaya mosaic virus in papaya plants (Carica papaya) in Mexico.

We report the results of a survey for the presence of Papaya ringspot virus (PRSV) along the coasts of the Gulf of Mexico and the Pacific Ocean, in 15 federal states of Mexico that account for over 98% of the national papaya production. More than 80 locations were visited in 58 counties. Out of a total of 267 papaya leaf samples, 157 tested positive for PRSV. We tested for the presence of three other viruses because of the occurrence of severe, atypical symptoms in plantations. Only Papaya mosaic virus (PapMV) was detected. PRSV was present in every county. PapMV was less frequent, but its overall distribution was almost identical. PRSV and PapMV occurred in single or mixed infections of papaya and other host species that could function as virus reservoirs. We investigated the diversity of the coat protein (CP) sequences of 36 PRSV isolates. The amino acid sequence divergence among all isolates ranged from 0.4 to 9.9%, and was comparable to that found in other regions of the world. In contrast to most of these world regions, there is a clear correlation between CP sequence variation and the geographical origins of the virus isolates.

The Absence of Eukaryotic Initiation Factor eIF(iso)4E Affects the Systemic Spread of a Tobacco etch virus Isolate in Arabidopsis thaliana

Translation initiation factor eIF4E exerts an important role during infection of viral species in the family Potyviridae. Particularly, a eIF(iso)4E family member is required for Arabidopsis thaliana susceptibility to Turnip mosaic virus, Lettuce mosaic virus, and Tobacco etch virus (TEV). In addition, a resistance mechanism named restriction of TEV movement (RTM) in A. thaliana controls the systemic spread of TEV in Col-0 ecotype. Here, we describe that TEV-TAMPS, a Mexican isolate, overcomes the RTM resistance mechanism reported for TEV-7DA infection of the Col-0 ecotype but depends on eIF(iso)4E for its systemic spread. To understand at which level eIF(iso)4E participates in A. thaliana TEV-TAMPS infection, the viral RNA replication and translation were measured. The absence or overexpression of eIF(iso)4E did not affect viral translation, and replication was still observed in the absence of eIF(iso)4E. However, the TEV-TAMPS systemic spread was completely abolished in the null mutant. The viral protein genome-linked (VPg) precursor NIa was found in coimmunoprecipitated complexes with both, eIF(iso)4E and eIF4E. However, the viral coat protein (CP) was only present in the eIF(iso)4E complexes. Since both the VPg and the CP proteins are needed for systemic spread, we propose a role of A. thaliana eIF(iso)4E in the movement of TEV-TAMPS within this host.

Coat protein gene sequence of a Mexican isolate of Sugarcane mosaic virus and its infectivity in maize and sugarcane plants

(SrMV) [2, 3]. Initially, viral isolates foundin sugarcane were designated as strains of SCMV and those found in maize asstrainsofMDMV,butbasedonreclassification,isolatesofSCMVandMDMVcanbe found in maize. Both SCMV and MDMV cause systemic mosaic symptomsand occasional stunting, and are not visually distinguishable in maize plants.The isolates can only be differentiated through nucleotide sequence comparison,specific serological reactions, or inoculation in differentials plants.

Virus-like particles from Escherichia Coli-derived untagged papaya ringspot virus capsid protein purified by immobilized metal affinity chromatography enhance the antibody response against a soluble antigen.

There is a growing interest in using virus-like particles (VLPs) as scaffolds for the presentation of antigens of choice to the immune system. In this work, VLPs from papaya ringspot virus capsid protein expressed in Escherichia coli were evaluated as enhancers of antibody response against a soluble antigen. Interestingly, although the capsid protein lacks a histidine tag, its purification by immobilized metal affinity chromatography was achieved. The formation of VLPs was demonstrated by electron microscopy for the first time for this capsid protein. VLPs were enriched by polyethylene glycol precipitation. Additionally, these VLPs were chemically coupled to green fluorescent protein in order to evaluate them as antigen carriers; however, bioconjugate instability was observed. Nonetheless, the adjuvant effect of these VLPs on BALB/c mice was evaluated, using GFP as antigen, resulting in a significant increase in anti-GFP IgG response, particularly, IgG1 class, demonstrating that the VLPs enhance the immune response against the antigen chosen in this study.

Antagonism or synergism between papaya ringspot virus and papaya mosaic virus in Carica papaya is determined by their order of infection.

Antagonism between unrelated plant viruses has not been thoroughly described. Our studies show that two unrelated viruses, papaya ringspot virus (PRSV) and papaya mosaic virus (PapMV) produce different symptomatic outcomes during mixed infection depending on the inoculation order. Synergism occurs in plants infected first with PRSV or in plants infected simultaneously with PRSV and PapMV, and antagonism occurs in plants infected first with PapMV and later inoculated with PRSV. During antagonism, elevated pathogenesis-related (PR-1) gene expression and increased reactive oxygen species production indicated the establishment of a host defense resulting in the reduction in PRSV titers. Polyribosomal fractioning showed that PRSV affects translation of cellular eEF1α, PR-1, β-tubulin, and PapMV RNAs in planta, suggesting that its infection could be related to an imbalance in the translation machinery. Our data suggest that primary PapMV infection activates a defense response against PRSV and establishes a protective relationship with the papaya host.

Immune response to a potyvirus with exposed amino groups available for chemical conjugation

BackgroundThe amino terminus of the tobacco etch virus (TEV) capsid protein is located on the external surface of infectious TEV particles, as proposed by previous studies and an in silico model. The epsilon amino groups on the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as antigen carriers. The availability of amino groups on the surfaces of TEV particles was determined and the immune response to TEV evaluated.ResultsUsing a biotin-tagged molecule that reacts specifically with amino groups, we found that the TEV capsid protein has amino groups on its surface available for coupling to other molecules via crosslinkers. Intraperitoneal TEV was administered to female BALB/c mice, and both their humoral and cellular responses measured. Different IgG isotypes, particularly IgG2a, directed against TEV were induced. In a cell proliferation assay, only spleen cells from vaccinated mice that were stimulated in vitro with TEV showed significant proliferation of CD3+/CD4+ and CD3+/CD8+ subpopulations and secreted significant amounts of interferon γ.ConclusionsTEV has surface amino groups that are available for chemical coupling. TEV induces both humoral and cellular responses when administered alone intraperitoneally to mice. Therefore, TEV should be evaluated as a vaccine adjuvant when chemically coupled to antigens of choice.

First Report of Leek yellow stripe virus in Garlic in the State of Guanajuato, Mexico

Garlic (Allium sativum L.) can be affected by a virus complex (1) consisting of two potyviruses, Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), and two carlaviruses, Garlic common latent virus (GCLV) and Shallot latent virus (SLV) (1). To identify the components of the virus complex that could be present in garlic plants in Guanajuato State, which is the second largest garlic producer in the country and where presumptive viral symptoms were initially observed in December 2004, a survey was carried out in six locations: San Miguel de Allende and San Luis de la Paz in northern Guanajuato; Irapuato and Villagrán in the central region; and Salamanca and Valle de Santiago in the southern part of the state. Enzyme-linked immunosorbent assay (ELISA) was carried out to detect LYSV, OYDV, GCLV, and SLV in 195 garlic leaf samples collected during January 2005 from plants with leaf yellow stripe, mosaic, enation, deformation, or dwarfism symptoms. A set of primers, previously reported and specific to the coat protein cistron of LYSV (1), were synthesized and used in a reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified product (1,020 nucleotides) was cloned into plasmid pGEM T-Easy (Promega, Madison, WI) and sequenced (Gen-bank Accession No. DQ841554). Sequence analysis showed that the cloned DNA fragment shared 97% similarity with the coat protein cistron of LYSV isolate no. 3 from Okinawa (GenBank Accession No. AB194632). The fragment was then radioactively labelled and used as a probe in the RNA blot analysis of all samples to confirm the ELISA results of LYSV. Of the 195 samples, 64 tested positive by RNA blot analysis. Forty-one of these were also positive by ELISA for LYSV. Preliminary, positive ELISA results were also obtained for OYDV, GCLV and SLV. To our knowledge, this is the first report of LYSV in the State of Guanajuato and in Mexico. The correct identification of viruses present in garlic will help to use the appropriate strategies to reduce viral incidence in this garlic-producing region. Reference: (1) T. V. M. Fajardo et al. Fitopatol. Bras. 26:619, 2001.

Highly efficient strategy for the heterologous expression and purification of soluble Cowpea chlorotic mottle virus capsid protein and in vitro pH-dependent assembly of virus-like particles.

Obtaining pure and soluble viral capsid proteins (CPs) has been a major challenge in the fields of science and technology in recent decades. In many cases, the CPs can self-assemble in the absence of a viral genome, resulting in non-infectious, empty virus-like particles (VLPs) which can be safely handled. The use of VLPs has found great potential in biotechnology and health purposes. In addition, VLPs are a good model system to study protein-protein interactions at the molecular level. In this work, an optimized strategy for the heterologous expression of the Cowpea chlorotic mottle virus (CCMV) CP based in Escherichia coli is described. The method is efficient, inexpensive and it consistently produces higher yields and greater purity levels than those reported so far. Additionally, one of the main advantages of this method is the prevention of the formation of inclusion bodies, thus allowing to directly obtain high amounts of the CP in a soluble and functionally active state with the capacity to readily form VLPs in vitro. The CCMV CP self-assembly pH dependence was also investigated, providing guidelines to easily modulate the process.

Porcine circovirus type 2 protective epitope densely carried by chimeric papaya ringspot virus–like particles expressed in Escherichia coli as a cost-effective vaccine manufacture alternative

Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus‐like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self‐assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N‐ and C‐termini and a predicted antigenic region located near the N‐terminus. Wild‐type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope‐presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small‐scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2.