Alphavirus Research Articles

Pseudotyping Lentiviral Vectors with Aura Virus Envelope Glycoproteins for DC-SIGN–Mediated Transduction of Dendritic Cells

Lentiviral vectors (LVs) pseudotyped with envelope proteins of alphaviruses have recently attracted considerable interest for their potential as gene delivery tools. We report the production of human immunodeficiency virus type 1 (HIV-1)-derived LVs pseudotyped with envelope glycoproteins derived from the Aura virus (AURA). We found that the AURA-glycoprotein-pseudotyped LVs use C-type lectins (DC-SIGN and L-SIGN) as attachment factors. These interactions with DC-SIGN are specific as determined by inhibition assays and appear to facilitate transduction through a pH-dependent pathway. AURA-pseudotyped LVs were used to transduce monocyte-derived dendritic cells (DCs) and the transduction was shown to be DC-SIGN mediated, as illustrated by competitive inhibition with DC-SIGN and L-SIGN antibodies and yeast mannan. Comparisons with LVs enveloped with glycoproteins derived from vesicular stomatitis virus and Sindbis virus suggest that AURA-glycoprotein-bearing LVs might be useful to genetically modify DCs for the study of DC biology and DC-based immunotherapy.

A phase I dose escalation trial of vaccine replicon particles (VRP) expressing prostate-specific membrane antigen (PSMA) in patients (pts) with castration-resistant prostate cancer (CRPC): Preliminary results.

140 Background: Prostate-specific membrane antigen (PSMA), a type II transmembrane protein overexpressed in prostate cells as they emerge into the androgen-independent state, remains a target to which vaccines with novel adjuvants or naked DNA has been directed. The PSMA-Viral Replicon Package (VRP) is a propagation-defective vector system from an attentuated strain of an alpha virus (VEE) encoding PSMA for use as a vaccine in prostate cancer. The PSMA gene is encoded by a subgenomic messenger RNA (mRNA), leading to high level expression of PSMA protein and presentation to the immune system.. We report preliminary results of a first in human trial using PSMA VRP to elicit immune responses targeting PSMA. Methods: Up to 18 pts with chemotherapy naïve CRPC metastatic to bone or soft tissue were permitted with 12 pts enrolled with ECOG status of 0 or 1. PSMA VRP was administered SC doses of either 0.9×107 IU or 0.36×108 IU for a total of 5 doses given on weeks 1, 4, 7, 10 and 18. Adverse events, PSA, circulating tumor cells, and clinical disease progression were assessed. Immune assessment was performed on patients' sera by ELISA and flow cytometry of serum binding to PSMA expressing tumor cells (3T3) and cellular (IFN-g EliSpot) components. Patients underwent imaging with bone and CAT scans every 3 months. Results: Three pts received 0.9×107 IU and 9 received 0.36×108 IU. Of these, 8 pts received all 5 vaccines. Four pts were taken off study for disease progression. One pt at the 0.36×108 continues on study and will be receiving his seventh vaccine. The demographic featuresof the 12 subjects enrolled were similar (mean age 69±11 yrs). No significant adverse events were noted; no aberrant laboratory abnormalities were associated with treatment. Weak humoral responses were observed in 1 patient in the 0.9×107 IU group and in 3 treated at 0.36×108. Conclusions: In this first-in-man evaluation, vaccination with PSMA VRP appeared to be well-tolerated but does not appear to show impact on disease progression. Further studies are ongoing to assess immune responses and identify potential pathways for enhancement of the PSMA VRP approach. [Table: see text]

A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus

In murine models of Venezuelan equine encephalitis virus (VEEV) infection, the neutralising monoclonal antibody 1A3B-7 has been shown to be effective in passive protection from challenge by the aerosol route with serogroups I, II and Mucambo virus (formally VEE complex subtype IIIA). This antibody is able to bind to all serogroups of the VEEV complex when used in ELISA and therefore is an excellent candidate for protein engineering in order to derive a humanised molecule suitable for therapeutic use in humans. A Complementarity Determining Region (CDR) grafting approach using human germline IgG frameworks was used to produce a panel of humanised variants of 1A3B-7, from which a single candidate molecule with retained binding specificity was identified. Evaluation of humanised 1A3B-7 (Hu1A3B-7) in in vitro studies indicated that Hu1A3B-7 retained both broad specificity and neutralising activity. Furthermore, in vivo experiments showed that Hu1A3B-7 successfully protected mice against lethal subcutaneous and aerosol challenges with VEEV strain TrD (serogroup I). Hu1A3B-7 is therefore a promising candidate for the future development of a broad-spectrum antiviral therapy to treat VEEV disease in humans.

Laboratory diagnosis of chikungunya virus: Do we really need it?

Chikungunya (CHIK) fever is a re-emerging Aedes mosquito-transmitted viral disease caused by CHIK virus belonging to the Togaviridae family of genus Alphavirus. The disease is almost self-limiting, occurs with characteristic triad of sudden onset fever, rash and arthritis. During the recent outbreak CHIKV was also found to cause long-term arthralgia, severe neurological disease and even fatalities. Although there are no antiviral or vaccines available for CHIKV, still there are several advantages to diagnose the infection. The present article provides an overview of various diagnostic modalities available and its significance by searching PubMed MeSH terms "Chikungunya virus" and "Diagnosis" for recent articles. The gold standard of CHIKV diagnosis is culture, yet requires facilities and skills. Highly sensitive and specific PCR assays for CHIKV have been developed, but the reagents and equipment are costly for widespread use. Serological diagnosis by detecting IgM antibody is most widely used as it is relatively cheaper and easier to perform. Disadvantages of antibody testing are cross-reactivity with other alpha viruses, cannot differentiate between recent past and acute infection, and its sensitivity varies in clinical settings. When tested for diagnosing acute CHIKV disease, sensitivities were just 4 to 22% and after 1 week rose to more than 80%. As most acutely infected patients seek medical attention within the first few days of illness, the ideal test should detect RNA or antigen. Therefore, the more realistic aim would be to develop a reliable antigen detection assay that could be used in rural areas, where CHIKV infection often occurs.

Isolation and Characterization of Sylvatic Mosquito-Borne Viruses in Trinidad: Enzootic Transmission and a New Potential Vector of Mucambo Virus

Mosquito surveillance was carried out in three forested regions of Trinidad during July 2007-March 2009. A total of 185,397 mosquitoes representing at least 46 species was collected, divided into pools of 1-50 mosquitoes according to species and sex, and screened for arboviruses using cytopathic effect assays on Vero cell monolayers. Eighty-five viruses were isolated, including members of the genera Alphavirus (Mucambo virus; MUCV) and Orthobunyavirus (Caraparu, Oriboca, Bimiti, and Wyeomyia viruses). Species of the Culex subgenus Melanoconion accounted for 56% of the total number of mosquitoes collected and 97% of the viruses isolated; Cx. (Mel.) portesi accounted for 92% of virus isolations. Our results also implicate for the first time Aedes (Ochlerotatus) hortator as a potential vector of MUCV. Phylogenetic analyses of 43 MUCV strains suggest population subdivision within Trinidad, consistent with the hypothesis of enzootic maintenance in localized rodent populations.

Vector-borne alpha virus infections

Vector-borne alpha virus infections

Molecular characterization of Chikungunya virus isolates from clinical samples and adult Aedes albopictus mosquitoes emerged from larvae from Kerala, South India

Chikungunya virus (CHIKV), an arthritogenic alphavirus, is transmitted to humans by infected Aedes (Ae.) aegypti and Ae.albopictus mosquitoes. In the study, reverse-transcription PCR (RT PCR) and virus isolation detected CHIKV in patient samples and also in adult Ae.albopictus mosquitoes that was derived from larvae collected during a chikungunya (CHIK) outbreak in Kerala in 2009. The CHIKV strains involved in the outbreak were the East, Central and South African (ECSA) genotype that had the E1 A226V mutation. The viral strains from the mosquitoes and CHIK patients from the same area showed a close relationship based on phylogenetic analysis. Genetic characterization by partial sequencing of non-structural protein 2 (nsP2; 378 bp), envelope E1 (505 bp) and E2 (428 bp) identified one critical mutation in the E2 protein coding region of these CHIKV strains. This novel, non-conservative mutation, L210Q, consistently present in both human and mosquito-derived samples studied, was within the region of the E2 protein (amino acids E2 200-220) that determines mosquito cell infectivity in many alpha viruses. Our results show the involvement of Ae. albopictus in this outbreak in Kerala and appearance of CHIKV with novel genetic changes. Detection of virus in adult mosquitoes, emerged in the laboratory from larvae, also points to the possibility of transovarial transmission (TOT) of mutant CHIKV strains in mosquitoes.

Cocirculation of Rio Negro Virus (RNV) and Pixuna Virus (PIXV) in Tucumán province, Argentina

Venezuelan equine encephalitis complex includes viruses considered emerging pathogens for humans and animals in the Americas. Two members of this complex have been detected previously in Argentina: Rio Negro Virus (RNV), detected in mosquitoes from Chaco province and rodents from Formosa province, and Pixuna Virus (PIXV), detected in mosquitoes from Chaco province. To carry out surveillance studies in other parts of the country, detection of a 195-bp fragment of alphaviruses by RT-nested PCR was performed in mosquito samples from San Miguel de Tucumán city. Four pools resulted positive and three were sequenced. Two amplicons grouped with RNV and one with PIXV. This is the first report of viral activity of members of the Venezuelan equine encephalitis complex in north-eastern Argentina.

Phylogeographic Structure and Evolutionary History of Sindbis Virus

Sindbis (SIN) virus, Alphavirus, is a mosquito-borne and bird-associated virus with large geographic distribution in the Old World. We investigated the genetic diversity of 59 SIN strains after limited sequencing of their E2 glycoprotein genes. The SIN strains showed maximal diversity of 22.2% at the amino acid (aa) level, and formed five tentative genotypes. The SIN-I genotype included strains from Europe and Africa. Strains from Australia and East Asia formed SIN-II and SIN-III with about 12% and 15% aa divergence from SIN-I. The only isolate from New Zealand was distinct, and constitutes the SIN-V genotype. Isolates from Azerbaijan and China formed genotype SIN-IV with 15.6%-19.1% aa divergence from SIN-I to III and SIN-V. Phylogenetic analyses indicated that Aura virus was present before the recombinant alphavirus lineage arose. This is consistent with a South American origin of the SIN complex, and argue for a spread in North America before reaching Asia and Australia, followed by westward radiation into Africa and Europe. High levels of sequence identities were observed for geographic regions belonging to the same north-south axis, whereas the east-west genetic exchange appears to be limited. The observed phylogeographic structure was confirmed by distinct aa patterns within two-thirds of the structural protein-coding region of SIN virus strains from Saudi Arabia, Asia, and Australia. The present geography of the five SIN genotypes and subclusters within SIN-I correlate with major bird migration patterns.

Enzootic Activity of Pixuna and Rio Negro Viruses (Venezuelan Equine Encephalitis complex) in a Neotropical Region of Argentina

Venezuelan Equine Encephalitis complex viruses cause epidemics and epizootics periodically in some regions of the Americas. In Argentina, only enzootic Rio Negro virus (AG80-663) (RNV) has been isolated. To survey and identify activity of viruses that belong to Venezuelan Equine Encephalitis complex in a template region of the country, a generic Alphavirus RT-Nested PCR was performed in 99 mosquito pools collected in Chaco province. Five pools were positive, and amplicons were sequenced: four of them clustered with RNV(AG80-663) and one with Pixuna virus. This is the first report of the circulation of Pixuna virus in Argentina, and it confirms enzootic and endemic activity of RNV(AG80-663) in neotropical regions of this country.

Human alpha and beta papillomaviruses use different synonymous codon profiles.

Human papillomaviruses use rare codons relative to their hosts. It has been theorized that this is a mechanism to allow the virus to escape immune surveillance. In the present study, we examined the codings of four major genes of 21 human alpha (mucosatropic) viruses and 16 human beta (cutaneous-tropic) viruses. We compared the codon usage of different genes from a given papillomavirus and also the same genes from different papillomaviruses. Our data showed that codon usage was not always uniform between two genes of a given papillomavirus or between the same genes of papillomaviruses from different genera. We speculate as to why this might be and conclude that codon usage in the papillomaviruses may not only play a role in facilitating escape from immune surveillance but may also underlie some of the unanswered questions in the papillomavirus field.

Ross River Virus Disease in a Traveler to Australia

A 42‐year‐old Singaporean man was admitted to the National University Hospital on November 29, 2005. He presented with a 2‐day history of fever, myalgias, severe arthralgias, and a rash. He had first noticed that his left ankle was swollen and painful on movement. A day later he developed a generalized non‐pruritic rash. There was no significant past medical history. He had traveled to western Australia from November 14 to 20, 2005 where he had mainly visited lakes, parks, and rivers in and around Perth. There was no travel to any developing country in the past 6 months. On clinical examination his left ankle was swollen, erythematous with periarticular tenderness. There was full range of movement. He had a generalized maculopapular rash which was more prominent over his lower limbs. Full blood count, urinalysis, liver enzymes, and serum creatinine were all normal. His C‐reactive protein (CRP) was raised to 17 mg/L (normal value: less than 10 mg/L) and his ALT was raised to 101 U/L (normal range: 5–60 IU/L), but all other liver parameters were normal. Dengue PCR and dengue serology were negative. Parvovirus and rubella serologies were negative. Because of his recent travel history to Australia and the triad of fever, arthritis, and rash, we considered various arboviral diseases, and in particular, alpha virus infections. Further screening for arboviral diseases was performed by the Queensland Health Pathology and Science Services in Brisbane, Australia. Ross River IgM [enzyme immunoassay (EIA)] was found to be reactive while Ross River IgG (EIA) was negative. All other investigations including testing for Barmah Forest were negative. Unfortunately, chikungunya PCR was not done routinely in 2005; however, chikungunya is epidemiologically highly unlikely in this case as local transmission of chikungunya had not been reported in 2005 either in Singapore or in Australia. Chikungunya was only imported … Corresponding Author: Annelies Wilder‐Smith, MD, PhD, DTM&H Director, Travellers' Screening and Vaccination Clinic, Associate Professor, Department of Medicine, National University Singapore, Singapore, 5 Lower Kent Ridge Road, Singapore 119074. E‐mail: epvws{at}pacific.net.sg

Emerging infections in Asia and its possible global effects.

Emerging infections in Asia and its possible global effects.

Isolation and phylogenetic analysis of Mucambo virus (Venezuelan equine encephalitis complex subtype IIIA) in Trinidad

In the 1950s and 1960s, alphaviruses in the Venezuelan equine encephalitis (VEE) antigenic complex were the most frequently isolated arboviruses in Trinidad. Since then, there has been very little research performed with these viruses. Herein, we report on the isolation, sequencing, and phylogenetic analyses of Mucambo virus (MUCV; VEE complex subtype IIIA), including 6 recently isolated from Culex (Melanoconion) portesi mosquitoes and 11 previously isolated in Trinidad and Brazil. Results show that nucleotide and amino acid identities across the complete structural polyprotein for the MUCV isolates were 96.6-100% and 98.7-100%, respectively, and the phylogenetic tree inferred for MUCV was highly geographically- and temporally-structured. Bayesian analyses suggest that the sampled MUCV lineages have a recent common ancestry of approximately 198 years (with a 95% highest posterior density (HPD) interval of 63-448 years) prior to 2007, and an overall rate of evolution of 1.28 x 10(-4) substitutions/site/yr.

Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice. Selected variants were then used to vaccinate larger cohorts of mice and their sera were assayed by both ELISA and by plaque reduction neutralization test (PRNT). Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge. Our results indicate that it is possible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using directed molecular evolution.

Culex nigripalpus Theobald (Diptera, Culicidae) feeding habit at the Parque Ecológico do Tietê, São Paulo, Brazil

The blood feeding of a population of Cx. nigripalpus from Parque Ecológico do Tietê (PET) was investigated using an indirect ELISA protocol. Mosquitoes were captured outside houses. Five hundred sixteen engorged females collected in a reforested area and 25 in an open area were tested. Rodents and dogs were the most common blood sources, accounting for approximately 65.3% of blood meals. Human blood was detected in 10.9%, dog blood in 26.1%, chicken blood in 2.4%, and rodent blood in 39.2% of the 541 insects tested. ELISA failed in identifying the blood sources of 233 engorged females, indicating that the mosquitoes may have fed on a host which was not tested. One hundred six individuals were positive for more than one host. The unweighted human blood index was 0.14 and the rodent/human, human/chicken, and dog/rodent feeding index values were 2.70, 1.51, and 1.33, respectively. Furthermore, rodents are defensive hosts for this haematophagous insect which looks for another host to complete blood-feeding. Considering that rodents are potential reservoirs for Mucambo virus and Saint Louis encephalitis virus and that Cx. nigripalpus feed on the blood of those mammals, we hypothesize that mosquito population in PET could participate in the transmission cycle of those arboviruses. Additionally, this species might be involved in the transmission of Dirofilaria immitis to dogs at this area.

Direct broad-range detection of alphaviruses in mosquito extracts

Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.

Chikungunya-type fever outbreak: some aspects related to this new epidemic in Jaffna district, northern Sri Lanka

Chikungunya, a disease caused by alpha virus and transmitted by Aedes mosquitoes, had seldom been heard by the people of Jaffna until early November 2006, although a chikungunya-type fever epidemic was reported in western Sri Lanka in 1965 [1-3]. While few samples have been confirmed for the presence of CHIK virus, widespread of chikungunya-type fever outbreaks have been reported from many parts of the country since September 2006. Similar outbreaks of CHIK virus have also been reported from many states of India including Tamil Nadu since December 2005 [4-6]. In Sri Lanka, Mannar district, which lies very close (32km) to Rameshwaram island of Tamil Nadu, was the area to first experience the fever outbreak. It has been estimated that the ongoing fever epidemic in Jaffna district has affected thousands of people between November and mid December 2006 (personal communicationDPDHS Office, Jaffna). It is difficult to establish the origin and mode of spread of CHIK fever to Jaffna because this epidemic occurred when the main land route (A-9) between Jaffna and the rest of the country remained closed since August 2006 owing to renewed armed conflict. Only sea and air transport facilities are available to facilitate public movement between Jaffna and the rest of the country. The closure of the main highway has hindered the availability of food and drugs in the market. Even the available items are sold for higher prices, creating an additional burden for ordinary people to meet daily requirements and raising concerns about nutrition and health. Although the disease has affected a large number of people, there are no facilities in Jaffna Teaching Hospital to diagnose CHIK fever. According to the health authorities in Jaffna, a few samples sent by air to the Medical Research Institute were confirmed for CHIK virus. A study was carried out under these circumstances in a highly affected village in Jaffna district to assess the severity of the fever as well as public attitudes and responses toward the epidemic. A structured questionnaire-based study was carried out in mid December 2006. Entire households in Inuvil-West (09°43’N:80°01’E), a residential rural area located 7km away from Jaffna city, were visited during this study. The suspected chikungunya-type fever cases were initially reported from this locality and almost the entire village was affected by the disease. There is a single Government Central Dispensary in addition to a privately owned hospital and a few small medical centers. The questionnaire was composed of multiple choice questions and an interview. There are four major parts: a) demographic information, b) symptoms and manner of treatment for chikungunya fever, c) knowledge about chikungunya, mosquitoes and mosquito-borne diseases, and d) attitude and practice to prevent mosquito bites. Basically the structured questionnaire was formulated to elucidate information on: monthly income, educational background, number of family members affected by fever, symptoms, manner of treatment (from government hospitals or private medical centers), recovery, how much was known about chikungunya, mosquitoes and their role in disease transmission, severity of the mosquito menace, mosquito biting time, and type of personal protective measures taken against mosquito bites. The questionnaires were written in Tamil (native language) and administered to heads of households from 0900 h to 1300 h. If no male head of household was available, a female head of household was interviewed. Larval surveys were also carried out in houses of all the respondents to obtain the following Aedes larval indices a) House Index (HI) the percentage of houses or premises with one or more habitats positive for Aedes larvae, b) Container Index (CI) the percentage of water holding containers positive for Aedes larvae, and c) Breteau Index (BI) the number of positive containers per 100 houses [7]. One hundred sixty two heads of households (67 males and 95 females), consisting 653 individuals, were interTropical Medicine and Health Vol. 35 No. 3, 2007, pp. 249-252 Copyright 2007 by The Japanese Society of Tropical Medicine

Alphavirus immunotherapy of NPM/ALK+ anaplastic large cell lymphoma in a mouse model

2574 Background: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\underline{\mathrm{A}}\) \end{document}naplastic \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\underline{\mathrm{L}}\) \end{document}arge \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\underline{\mathrm{C}}\) \end{document}ell \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\underline{\mathrm{L}}\) \end{document}ymphoma (ALCL) is a non-Hodgkin's lymphoma. 2/3 of Kit+ (CD30) ALCL cases are associated with the expression of the fusion oncogene, NPM/ALK. Due to the effective immunogenicity of Venezuelan ...

Multiplex nested PCR for Brazilian Alphavirus diagnosis

A reverse-transcriptase PCR (RT–PCR) and a multiplex nested PCR were developed for the rapid detection and identification of 14 Brazilian alphaviruses. Using Alphavirus genus-specific primers in a RT–PCR, we obtained amplified products of 434 bp. Species-specific primers were selected and simultaneously tested in a multiplex nested PCR. The nested PCR increased the test sensitivity 1000-fold and was capable of identifying Brazilian Alphavirus showing the expected bands with diagnostic sizes for Venezuelan (400 bp), Eastern (124 bp), and Western (208 bp) equine encephalitis, Aura (86 bp), and Mayaro (270 bp) viruses. This strategy for diagnosis is fast, sensitive, specific and it can be used as a reliable alternative for routine Brazilian Alphavirus diagnosis.