Two new begomoviruses infecting tomato and Hibiscus sp. in the Amazon region of Brazil.

Two begomoviruses were isolated in the northern Brazilian state of Pará, infecting non-cultivated Hibiscus sp. and cultivated tomato (Solanum lycopersicum). The complete genomes (DNA-A and DNA-B) of the two viruses showed the typical organization of New World bipartite begomoviruses. Based on the species assignment criteria in the genus Begomovirus, each virus is a member of a new species. The virus from Hibiscus is most closely related to sida yellow mosaic Yucatan virus, while the tomato virus is most closely related to abutilon mosaic Brazil virus and corchorus mottle virus. Recombination events were detected in the DNA-A of the tomato virus, but not in the Hibiscus virus genome. We propose the names "hibiscus golden mosaic virus" (HGMV) and "tomato chlorotic leaf curl virus" (ToCLCV) for the viruses reported in this study.

Detection of Mesta yellow vein mosaic virus (MeYVMV) in field samples by a loop-mediated isothermal amplification reaction.

A loop-mediated isothermal amplification (LAMP) assay was optimized for the detection of Mesta yellow vein mosaic virus (MeYVMV) in diseased plants of mesta (Hibiscus sabdariffa L.& H. cannabinus L.). The LAMP assay was optimized using a set of six primers targeting the MeYVMV genome and could be completed in 30-60 min at 63 °C. The LAMP amplification results were visualized by adding 1 µl of hydroxy naphthol blue (HNB) dye in a 25 µl LAMP reaction mixture prior to amplification as well as by electrophoresis. The LAMP assay, which detected MeYVMV in a 10-5-fold diluted total DNA, was more sensitive than the PCR assay (10-4-fold dilution). The optimized LAMP assay was able to detect MeYVMV in different parts of the kenaf and roselle plants. Similarly, the optimized PCR assay was also capable of detecting MeYVMV in all the different parts of the kenaf plant but failed to detect the virus in the stem and flower buds of the roselle plant. Validation of the LAMP and LAMP with HNB dye assays revealed that the optimized reactions can be used successfully for the in-situ detection of MeYVMV in field samples and in virus quarantine programs. This is the first report of the detection of the begomovirus species, MeYVMV, in the mucilaginous plant species, kenaf and roselle, using a LAMP assay.

Unveiling the complete genome sequence of clerodendrum chlorotic spot virus, a putative dichorhavirus infecting ornamental plants.

The genus Dichorhavirus includes plant-infecting rhabdoviruses with bisegmented genomes that are horizontally transmitted by false spider mites of the genus Brevipalpus. The complete genome sequences of three isolates of the putative dichorhavirus clerodendrum chlorotic spot virus were determined using next-generation sequencing (Illumina) and traditional RT-PCR. Their genome organization, sequence similarity and phylogenetic relationship to other viruses, and transmissibility by Brevipalpus yothersi mites support the assignment of these viruses to a new species of dichorhavirus, as suggested previously. New data are discussed stressing the reliability of the current rules for species demarcation and taxonomic status criteria within the genus Dichorhavirus.

Identification and Characterization of Citrus Chlorotic Spot Virus, a New Dichorhavirus Associated with Citrus Leprosis-Like Symptoms.

Local chlorotic spots resembling early lesions characteristic of citrus leprosis (CL) were observed in leaves of two sweet orange (Citrus sinensis L.) trees in Teresina, State of Piauí, Brazil, in early 2017. However, despite the similarities, these spots were generally larger than those of a typical CL and showed rare or no necrosis symptoms. In symptomatic tissues, transmission electron microscopy revealed the presence of viroplasms in the nuclei of the infected parenchymal cells and rod-shaped particles with an average size of approximately 40 × 100 nm, resembling those typically observed during infection by dichorhaviruses. A bipartite genome of the putative novel virus, tentatively named citrus chlorotic spot virus (CiCSV) (RNA1 = 6,518 nucleotides [nt] and RNA2 = 5,987 nt), revealed the highest nucleotide sequence identity values with the dichorhaviruses coffee ringspot virus strain Lavras (73.8%), citrus leprosis virus N strain Ibi1 (58.6%), and orchid fleck virus strain So (56.9%). In addition to citrus, CiCSV was also found in local chlorotic lesions on leaves of the ornamental plant beach hibiscus (Talipariti tiliaceum (L.) Fryxell). Morphological characterization of mites recovered from the infected plants revealed at least two different types of Brevipalpus. One of them corresponds to Brevipalpus yothersi. The other is slightly different from B. yothersi mites but comprises traits that possibly place it as another species. A mix of the two mite types collected on beach hibiscus successfully transmitted CiCSV to arabidopsis plants but additional work is required to verify whether both types of flat mite may act as viral vectors. The current study reveals a newly described dichorhavirus associated with a citrus disease in the northeastern region of Brazil.

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

The length of an internal poly(A) tract of hibiscus latent Singapore virus is crucial for its replication.

Hibiscus latent Singapore virus (HLSV) mutants were constructed to study roles of its internal poly(A) tract (IPAT) in viral replication and coat protein (CP) expression. Shortening of the IPAT resulted in reduced HLSV RNA accumulation and its minimal length required for HLSV CP expression in plants was 24 nt. Disruption of a putative long range RNA-RNA interacting structure between 5' and 3' untranslated regions of HLSV-22A and -24A resulted in reduced viral RNA and undetectable CP accumulation in inoculated leaves. Replacement of the IPAT in HLSV with an upstream pseudoknot domain (UPD) of Tobacco mosaic virus (TMV) or insertion of the UPD to the immediate downstream of a 24 nt IPAT in HLSV resulted in drastically reduced viral RNA replication. Plants infected with a TMV mutant by replacement of the UPD with 43 nt IPAT exhibited milder mosaic symptoms without necrosis. We have proposed a model for HLSV replication.

Hibiscus chlorotic ringspot virus coat protein is essential for cell-to-cell and long-distance movement but not for viral RNA replication.

Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus L.), two HCRSV mutants, designated as p2590 (A to G) in which the first start codon ATG was replaced with GTG and p2776 (C to G) in which proline 63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G) were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for ß-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G) was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G) inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the ß-annulus domain is required in T = 3 assembly in vitro.

Complete nucleotide sequence and genome structure of a Japanese isolate of hibiscus latent Fort Pierce virus, a unique tobamovirus that contains an internal poly(A) region in its 3' end.

In this study, we detected a Japanese isolate of hibiscus latent Fort Pierce virus (HLFPV-J), a member of the genus Tobamovirus, in a hibiscus plant in Japan and determined the complete sequence and organization of its genome. HLFPV-J has four open reading frames (ORFs), each of which shares more than 98 % nucleotide sequence identity with those of other HLFPV isolates. Moreover, HLFPV-J contains a unique internal poly(A) region of variable length, ranging from 44 to 78 nucleotides, in its 3'-untranslated region (UTR), as is the case with hibiscus latent Singapore virus (HLSV), another hibiscus-infecting tobamovirus. The length of the HLFPV-J genome was 6431 nucleotides, including the shortest internal poly(A) region. The sequence identities of ORFs 1, 2, 3 and 4 of HLFPV-J to other tobamoviruses were 46.6-68.7, 49.9-70.8, 31.0-70.8 and 39.4-70.1 %, respectively, at the nucleotide level and 39.8-75.0, 43.6-77.8, 19.2-70.4 and 31.2-74.2 %, respectively, at the amino acid level. The 5'- and 3'-UTRs of HLFPV-J showed 24.3-58.6 and 13.0-79.8 % identity, respectively, to other tobamoviruses. In particular, when compared to other tobamoviruses, each ORF and UTR of HLFPV-J showed the highest sequence identity to those of HLSV. Phylogenetic analysis showed that HLFPV-J, other HLFPV isolates and HLSV constitute a malvaceous-plant-infecting tobamovirus cluster. These results indicate that the genomic structure of HLFPV-J has unique features similar to those of HLSV. To our knowledge, this is the first report of the complete genome sequence of HLFPV.

An infectious RNA with a hepta-adenosine stretch responsible for programmed -1 ribosomal frameshift derived from a full-length cDNA clone of Hibiscus latent Singapore virus.

Hibiscus latent Singapore virus (HLSV) is a member of Tobamovirus and its full-length cDNA clones were constructed. The in vitro transcripts from two HLSV full-length cDNA clones, which contain a hepta-adenosine stretch (pHLSV-7A) and an octo-adenosine stretch (pHLSV-8A), are both infectious. The replication level of HLSV-7A in Nicotiana benthamiana protoplasts was 5-fold lower, as compared to that of HLSV-8A. The replicase proteins of HLSV-7A were produced through programmed -1 ribosomal frameshift (-1 PRF) and the 7A stretch was a slippery sequence for -1 PRF. Mutations to the downstream pseudoknot of 7A stretch showed that the pseudoknot was not required for the frameshift in vitro. The stretch was found to be extended to 8A after subsequent replication cycles in vivo. It is envisaged that HLSV employs the monotonous runs of A and -1 PRF to convert its 7A to 8A to reach higher replication for its survival in plants.

A method of direct PCR without DNA extraction for rapid detection of begomoviruses infecting jute and mesta.

UNLABELLED: We have developed a simple method of direct PCR (dPCR) without time-consuming procedures of DNA extraction by directly using the leaf bits for rapid detection of begomoviruses in jute and mesta. The leaf bits were treated with a lysis buffer for 35 min, and the lysate was used as PCR template. Different components and their concentration in lysis buffer systems were optimized and the optimal buffer system composed of 20 mmol l(-1) tris (hydroxymethyl aminomethane (Tris)-Cl (pH 8·0), 1·5 mmol l(-1) ethylenediaminetetraacetic acid (EDTA) (pH 8·0), 1·4 mol l(-1) NaCl and 200 µg/mL Proteinase K. Further, 3% PVP (w/v) and ß-marcaptoethanol (1% v/v) were additionally added into the buffer in case of jute. Under optimized PCR conditions, both viral DNA as well as plant (jute and mesta) genomic DNA were amplified from the lysate. dPCR required fewer reagents and less incubation time reducing both time and cost of detection. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of begomoviruses by serology is not suitable due to difficulty in preparing high titre and specific antisera. Begomoviruses are routinely detected by PCR-based techniques using universal or specific primers. However, it is a prerequisite to isolate pure DNA from the samples before PCR. DNA extraction from some plants such as jute, mesta is very difficult due to the presence of mucilage and other impurities. Therefore, we have developed a method of direct PCR without DNA extraction for detection of begomoviruses from these crops. It is the first report of a direct PCR method in jute and mesta.

Basic amino acid mutations in the nuclear localization signal of hibiscus chlorotic ringspot virus p23 inhibit virus long distance movement.

The p23 is a unique protein in the Hibiscus chlorotic ringspot virus which belongs to Family Tombusviridae Genus Carmovirus. Our previous results showed that the p23 is indispensable for host-specific replication and is localized in the nucleus with a novel nuclear localization signal. To investigate additional function(s) of p23, mutations of basic amino acids lysine (K), arginine (R) and histidine (H) that abolish its nuclear localization, were introduced into a biologically active full-length cDNA clone p223 of HCRSV for testing its effects on virus replication and virus movement in vivo. Primer-specific reverse transcription-PCR was conducted to detect gene transcript level of p23 and viral coat protein separately. Virus replication and its coat protein expression were detected by fluorescent in situ hybridization and Western blot, respectively. The effect of p23 was further confirmed by using artificial microRNA inoculation-mediated silencing. Results showed that the two mutants were able to replicate in protoplasts but unable to move from inoculated leaves to newly emerged leaves. Both the p23 and the CP genes of HCRSV were detected in the newly emerged leaves of infected plants but CP was not detected by Western blot and no symptom was observed on those leaves at 19 days post inoculation. This study demonstrates that when p23 is prevented from entering the nucleus, it results in restriction of virus long distance movement which in turn abrogates symptom expression in the newly emerged leaves. We conclude that the p23 protein of HCRSV is required for virus long distance movement.

A cilevirus infects ornamental hibiscus in Hawaii.

The complete nucleotide sequence of a virus infecting ornamental hibiscus (Hibiscus sp.) in Hawaii with symptoms of green ringspots on senescing leaves was determined from double-stranded RNA isolated from symptomatic tissue. Excluding polyadenylated regions at the 3' termini, the bipartite RNA genome was 8748 and 5019 nt in length for RNA1 and RNA2, respectively. The genome organization was typical of a cilevirus: RNA1 encoded a large replication-associated protein with methyltransferase, protease, helicase and RNA-dependent RNA polymerase domains as well as a 29-kDa protein of unknown function. RNA2 possessed five open reading frames that potentially encoded proteins with molecular masses of 15, 7, 62, 32, and 24 kDa. The 32-kDa protein is homologous to 3A movement proteins of RNA viruses; the other proteins are of unknown function. A proteome comparison revealed that this virus was 92 % identical to citrus leprosis virus cytoplasmic type 2 (CiLV-C2), a recently characterized cilevirus infecting citrus with leprosis-like symptoms in Colombia. The high sequence similarity suggests that the virus described in this study could be a strain of CiLV-C2, but since the new genus Cilevirus does not have species demarcation criteria established at present, the classification of this virus infecting hibiscus is open to interpretation. This study represents the first documented case of a cilevirus established in the United States and provides insight into the diversity within the genus Cilevirus.

Plant growth retardation and conserved miRNAs are correlated to Hibiscus chlorotic ringspot virus infection.

Virus infection may cause a multiplicity of symptoms in their host including discoloration, distortion and growth retardation. Hibiscus chlorotic ringspot virus (HCRSV) infection was studied using kenaf (Hibiscus cannabinus L.), a non-wood fiber-producing crop in this study. Infection by HCRSV reduced the fiber yield and concomitant economic value of kenaf. We investigated kenaf growth retardation and fluctuations of four selected miRNAs after HCRSV infection. Vegetative growth (including plant height, leaf size and root development) was severely retarded. From the transverse and radial sections of the mock and HCRSV-infected kenaf stem, the vascular bundles of HCRSV-infected plants were severely disrupted. In addition, four conserved plant developmental and defence related microRNAs (miRNAs) (miR165, miR167, miR168 and miR171) and their respective target genes phabulosa (PHB), auxin response factor 8 (ARF8), argonaute 1 (AGO1) and scarecrow-like protein 1 (SCL1) displayed variation in expression levels after HCRSV infection. Compared with the mock inoculated kenaf plants, miR171 and miR168 and their targets SCL1 and AGO1 showed greater fluctuations after HCRSV infection. As HCRSV upregulates plant SO transcript in kenaf and upregulated AGO1 in HCRSV-infected plants, the expression level of AGO1 transcript was further investigated under sulfite oxidase (SO) overexpression or silencing condition. Interestingly, the four selected miRNAs were also up- or down-regulated upon overexpression or silencing of SO. Plant growth retardation and fluctuation of four conserved miRNAs are correlated to HCRSV infection.

Indexing of yellow vein mosaic disease of mesta (Hibiscus cannabinus).

The assessment of severity with chronological development of disease of mesta (Hibiscus cannabinus, cv. HC-583) on a particular diseased plant was studied. Based on the symptomatology and intensity of diseases caused by Begomovirus an empirical formula was developed. A methodology for assessing the disease intensity of yellow vein mosaic disease of mesta grown under natural field condition was proposed and it was validated by comparing with infected plants grown under field as well as glasshouse conditions. Quantitative assessment of the disease indicated that the disease index value at different stages of pathogenesis varied from 1.82 to 49.44 under field condition whereas the same varied from 2.78 to 50.83 under glasshouse condition. Mortality of infected leaves was noticed when the index value reached maximum. With the present methodology scoring of gradual development of disease has become possible for a particular plant and even for an individual leaf of a particular cultivar.

Identification of a plant viral RNA genome in the nucleus.

Viruses contain either DNA or RNA as genomes. DNA viruses replicate within nucleus, while most RNA viruses, especially (+)-sense single-stranded RNA, replicate and are present within cytoplasm. We proposed a new thought that is contrary to the common notion that (+)-sense single-stranded RNA viruses are present only in the cytoplasm. In this study, we question whether the genome of a plant RNA virus (non-retroviral) is present in the nucleus of infected cells? Hibiscus chlorotic ringspot virus (HCRSV) RNA was detected in the nucleus of infected cells, as shown by fluorescent in situ hybridization. Western blot using anti-histone 3 and anti-phosphoenolpyruvate carboxylase showed that nuclei were highly purified from mock and HCRSV-infected kenaf (Hibiscus cannabilis L.) leaves, respectively. The p23 and HCRSV coat protein (CP) coding regions were both amplified from total RNA extracted from isolated nuclei. Viral RNA in the nucleus may be used to generate viral microRNAs (vir-miRNAs), as five putative vir-miRNAs were predicted from HCRSV using the vir-miRNAs prediction database. The vir-miRNA (hcrsv-miR-H1-5p) was detected using TaqMan® stem-loop real-time PCR, and by northern blot using DIG-end labeled probe in HCRSV-infected kenaf leaves. Finally, a novel nuclear localization signal (NLS) was discovered in p23 of HCRSV. The NLS interacts with importin α and facilitates viral RNA genome to enter nucleus. We demonstrate the presence of a (+)-sense single-stranded viral RNA within nucleus.

Hibiscus chlorotic ringspot virus coat protein upregulates sulfur metabolism genes for enhanced pathogen defense.

In both Hibiscus chlorotic ringspot virus (HCRSV)-infected and HCRSV coat protein (CP) agroinfiltrated plant leaves, we showed that sulfur metabolism pathway related genes-namely, sulfite oxidase (SO), sulfite reductase, and adenosine 5'-phosphosulfate kinase-were upregulated. It led us to examine a plausible relationship between sulfur-enhanced resistance (SED) and HCRSV infection. We broadened an established method to include different concentrations of sulfur (0S, 1S, 2S, and 3S) to correlate them to symptom development of HCRSV-infected plants. We treated plants with glutathione and its inhibitor to verify the SED effect. Disease resistance was induced through elevated glutathione contents during HCRSV infection. The upregulation of SO was related to suppression of symptom development induced by sulfur treatment. In this study, we established that HCRSV-CP interacts with SO which, in turn, triggers SED and leads to enhanced plant resistance. Thus, we have discovered a new function of SO in the SED pathway. This is the first report to demonstrate that the interaction of a viral protein and host protein trigger SED in plants. It will be interesting if such interaction applies generally to other host-pathogen interactions that will lead to enhanced pathogen defense.

Characterization of a virus infecting Citrus volkameriana with citrus leprosis-like symptoms.

A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves but not in healthy controls. Double-stranded (ds) RNAs ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay; however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.

Biochemical characterization of compatible plant-viral interaction: a case study with a begomovirus-kenaf host-pathosystem.

Yellow vein mosaic disease of mesta, a compatible plant virus interaction poses a serious threat to mesta cultivation in India. Plants respond to invasion by pathogens with multi component defense responses particularly in incompatible interaction. With the aim of understanding, a biochemical approach was attempted to study the cellular redox status in early stages of yellow vein mosaic virus infection associated with different age's plant of Hibiscus cannabinus. Comparative analysis of GSH and GSSG content in infected and control plant of different ages indicated that infected plants are under oxidative or nitrosative stress condition. A significant change was observed in Glutathione Reductase, Catalase and Ascorbate Peroxidase level in early stage of infection. We also showed microscopic evidence of nitrosylated thiols in infected leaves, stems and roots of H. cannabinus. Furthermore, we identified few defense related proteins in infected plant using MALDI TOF mass spectrometric analysis.

Structure of hibiscus latent singapore virus by fiber diffraction: a nonconserved his122 contributes to coat protein stability.

Hibiscus latent Singapore virus (HLSV) is a rigid rod-shaped plant virus and a new member of the Tobamovirus family. Unlike all other Tobamoviruses, the HLSV genome contains a unique poly(A) tract in its 3' untranslated region. The virion is composed of a monomeric coat protein (CP) unit of 18 kDa, arranged as a right-handed helix around the virus axis. We have determined the structure of HLSV at 3.5 Å by X-ray fiber diffraction and refined it to an R-factor of 0.096. While the overall structure of the HLSV CP resembles that of other Tobamoviruses, there are a few unique differences. There is a kink in the LR helix due to the presence of His122. Also, the adjacent Lys123 may further destabilize the helix by positive charge repulsion, making the kink more pronounced. The His122-Asp88 salt bridge provides significant stability to the loop adjacent to the RR helix. Carboxyl-carboxylate interactions that drive viral disassembly are also different in HLSV. The nucleotide recognition mechanisms for virus assembly between HLSV and ribgrass mosaic virus are similar, but different between tobacco mosaic virus and cucumber green mottle mosaic virus.

Production of nitric oxide in host-virus interaction: a case study with a compatible Begomovirus-Kenaf host-pathosystem.

Nitric oxide (NO) plays a key role in plant diseases resistance. Here we have first time demonstrated that begomovirus infection in susceptible H. cannabinus plants, results in elevated NO and reactive nitrogen species production during early infection stage not only in infected leaf but also in root and shoot. Production of NO was further confirmed by oxyhemoglobin assay. Furthermore, we used Phenyl alanine ammonia lyase as marker of pathogenesis related enzyme. In addition evidence for protein tyrosine nitration during the early stage of viral infection clearly showed the involvement of nitrosative stress.