Vesicular Virus Research Articles

Concomitant Syndromic Diagnosis of Mpox and Other Vesicular Viruses in Patients with Skin and Genital Lesions.

The recent multi-country outbreak of the zoonotic monkeypox virus (MPXV) infection in humans without an epidemiological link with endemic areas has raised concerns about the route of transmission. Since the infection spread largely among men who have sex with men who, in most cases, presented primary lesions of the genital and oral mucosa, sexual transmission has been proposed. In the present study, we retrospectively evaluated specimens of vesicular lesions collected from the skin and genital tract of 35 patients (23 positive and 12 negative) presenting at our Institute for monkeypox (mpox) diagnosis by using a novel molecular syndromic vesicular virus panel (VVP) assay. All MPXV-positive samples but one was confirmed; however, the viral syndromic analysis revealed that 8.6% of them were coinfected with one or more viruses, and 17% had at least a virus different from the MPXV. The percentage of coinfections increased to more than 25% when nonviral pathogens, such as gonorrhea and syphilis, were also considered. These results show the usefulness of syndromic diagnosis in cases where MPXV is suspected (and vice versa) and at the same time highlight that the broader screening of sexually transmitted infections in the population with high-risk sexual behavior is critical to ensure a complete etiology and appropriate treatment.

Elimination of Non-cytopathic Bovine Viral Diarrhea Virus From the LFBK-αvβ6 Cell Line.

The LFBK-αvβ6 cell line is highly sensitive for the isolation of foot-and-mouth disease virus (FMDV) and porcinophilic vesicular viruses. However, LFBK-αvβ6 cells are contaminated with a non-cytopathic bovine viral diarrhea virus (BVDV), which complicates handling procedures in areas where other cell lines are maintained, as well downstream use of viral isolates. In this study, we used an aromatic cationic compound (DB772) to treat LFBK-αvβ6 cells using an approach that has been previously used to eliminate persistent BVDV from fetal fibroblast cell lines. After three cell passages with 4 μM DB772, BVDV could no longer be detected in unclarified cell suspensions using a pan-pestivirus real-time RT-PCR assay, and remained undetectable after treatment was stopped (nine passages) for an additional 28 passages. The analytical sensitivity of the DB772-treated LFBK-αvβ6 cultures (renamed WRL-LFBK-αvβ6) to titrations of FMDV and other vesicular virus isolates was comparable to untreated LFBK-αvβ6 cells. These new BVDV-free cells can be handled without the risk of cross-contaminating other cells lines or reagents, and used for routine diagnostics, in vivo studies and/or preparation of new vaccine strains.

Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus

This study describes the first multiway comparison of portable isothermal assays for the detection of foot-and-mouth disease virus (FMDV), benchmarked against real-time reverse transcription RT-PCR (rRT-PCR). The selected isothermal chemistries included reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). The analytical sensitivity of RT-LAMP was comparable to rRT-PCR (101 RNA copies), while RT-RPA was one log10 less sensitive (102 RNA copies). Diagnostic performance was evaluated using a panel of 35 samples from FMDV-positive cattle and eight samples from cattle infected with other vesicular viruses. Assay concordance for RT-LAMP and RT-RPA was 86–98% and 67–77%, respectively, when compared to rRT-PCR, with discordant samples consistently having high rRT-PCR cycle threshold values (no false-positives were detected for any assay). In addition, a hierarchy of sample preparation methods, from robotic extraction to simple dilution of samples, for epithelial suspensions, serum and oesophageal-pharyngeal (OP) fluid were evaluated. Results obtained for RT-LAMP confirmed that FMDV RNA can be detected in the absence of RNA extraction. However, simple sample preparation methods were less encouraging for RT-RPA, with accurate results only obtained when using RNA extraction. Although the evaluation of assay performance is specific to the conditions tested in this study, the compatibility of RT-LAMP chemistry with multiple sample types, both in the presence and absence of nucleic acid extraction, provides advantages over alternative isothermal chemistries and alternative pen-side diagnostics such as antigen-detection lateral-flow devices. These characteristics of RT-LAMP enable the assay to be performed over a large diagnostic detection window, providing a realistic means to rapidly confirm positive FMD cases close to the point of sampling.

Abnormal Morphological Vesicles in Influenza a Virus Exposed to Acid pH

Vesicles on the virion surface, which continued the lipoprotein membrane but had no spikes of virus glycoproteins hemagglutinin (HA) and neuraminidase (NA), were detected. These vesicles and virus particles were 18±7 and 103±12 nm in diameter, respectively, and, as a rule, one vesicle was found per virion. The locus of the external protrusion in the virion presumably corresponded to the site of virus budding during assembly in infected cell free from HA and NA spikes outside and M1 matrix protein inside, but enriched with ionic channel protein M2. Particles with vesicles constituted ~3-10% of the virus population produced in MDCK-H culture and containing uncleaved HA0 hemagglutinin. The content of vesicular virions increased slightly after trypsin cleavage HA0→HA1+HA2 and reached 10-15%. Exposure of the virus in acid medium (pH 4.3) led to a drastic increase of vesicular virions - to 60-80% for HA0 and HA1+HA2 virus. This was paralleled by changes in contrast permeability (for phosphotungstic acid). HA0 virions remained contrast-impermeable, while HA1+HA2 particles let the contrast in through the vesicles detected on the surface and were rapidly destroyed after incubation in acid medium. Hence, cleavage of the surface glycoprotein HA0 into HA1 and HA2 stimulated the acid-dependent permeability of the lipid membrane and led to attenuation of the ribonucleoprotein and protein matrix M1 contacts inside the virion.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6 TCID 50/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

The eIF2α kinases inhibit vesicular stomatitis virus replication independently of eIF2 phosphorylation.

The eIF2alpha kinases have been involved in the inhibition of vesicular virus replication but the contribution of each kinase to this process has not been fully investigated. Using mouse embryonic fibroblasts (MEFs) from knock-out mice we show that PKR and HRI have no effects on VSV replication as opposed to PERK and GCN2, which exhibit strong inhibitory effects. When MEFs containing the serine 51 to alanine mutation of eIF2alpha were used, we found that VSV replication is independent of eIF2alpha phosphorylation. Nevertheless, the kinase domain of the eIF2alpha kinases is both necessary and sufficient to inhibit VSV replication in cultured cells. Induction of PI3K-Akt/PKB pathway by eIF2alpha kinase activation plays no role in the inhibition of VSV replication. Our data provide strong evidence that VSV replication is not affected by eIF2alpha phosphorylation or downstream effector pathways such as the PI3K-Akt/PKB pathway. Thus, the anti-viral properties of eIF2alpha kinases are not always related to their inhibitory effects on host protein synthesis as previously thought and are possibly mediated by phosphorylation of proteins other than eIF2alpha.

Determination of interferon beta neutralizing antibodies in multiple sclerosis: Improvement of clinical sensitivity of a cytopathic effect assay

IntroductionNeutralizing antibodies (NAb) against interferon beta (IFNβ) reduce treatment efficacy in patients with multiple sclerosis (MS). ObjectiveWe used the cross-reactivity of NAbs against IFNβ-1a or IFNβ-1b for improving the sensitivity of NAb measurement. Patients/methodsThe study included sera from 185 MS patients treated at least 12 months (T1) with IFNβ-1a (Rebif®; n=62 or Avonex®; n=61) or IFNβ-1b (Betaferon®; n=62). NAbs were measured by CPE in all the sera using the WISH cell line infected by the bovine stomatitis vesicular virus. NAb titres were expressed in ten-fold reduction (TRU)/mL. NAb-positive titres at T1 were also measured 3 to 6 months later (T2). ResultsAt T1, with the classical CPE assay using the IFNβ type (1a or 1b) according to the molecule administered, 29/180 (15.7%) patients had positive NAb titres: 9/62 (14.5%), 14/62 (22.6%) and 6/61 (9.8%) subjects were treated with Betaferon®, Rebif® and Avonex®, respectively. When IFNβ-1a (Rebif® or Avonex®) was used, NAb-positive results were found in 33/185 (17.8%) patients. They included the 29 patients previously found positive with the classical CPE method and 7 other subjects treated with Betaferon® and NAb-negative against IFNβ-1b. All the 33 NAb-positive patients at T1 were positive 3 to 6 months later. ConclusionIFNβ-1a should be used for the NAb determination for an optimal evaluation of NAb-positive patients in MS patients treated with IFNβ.

Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses

A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.

Rapid and differential diagnosis of foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis by a new multiplex RT-PCR assay

A highly sensitive and specific one-step multiplex RT-PCR assay has been developed and standardised for the simultaneous and differential detection of the most important vesicular viruses affecting livestock: foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV). The method uses three primer sets, each one specific for the corresponding virus, selected to detect of all serotypes of FMD and VS. The detection range was confirmed by examination of a collection of 31 isolates of the three target viruses. The specificity of the assay was also demonstrated by testing other related viruses, uninfected cell line cultures and healthy pig tissues. The testing of blood and serum samples from animals infected experimentally proved that the method can be useful for early diagnosis of the diseases, even before the first vesicular lesions are visualized in the infected pigs. An assessment of the performance of the multiplex RT-PCR was carried out using a panel of more than 100 samples from animals infected experimentally, showing the suitability of the method for a rapid (less than 6h), sensitive and specific differential diagnosis in clinical samples. Additionally, a uniplex RT-PCR for VSV, that amplifies the two viral serotypes, was also developed and tested as a rapid tool for the diagnosis of this vesicular disease.

Detection and Serotype-Specific Differentiation of Vesicular Stomatitis Virus Using a Multiplex, Real-Time, Reverse Transcription-Polymerase Chain Reaction Assay

A multiplex, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that allowed simultaneous detection and rapid differentiation of vesicular stomatitis virus strains--New Jersey (VSV-NJ) and Indiana 1, 2, and 3 (VSV-IN1-3). This assay involves use of a set of VSV universal primers located in the L gene that amplify VSV-IN1-3 and VSV-NJ using probes that allow differentiation of the major serotypes Indiana and New Jersey. The assay was evaluated using reference VSV, foot-and-mouth disease virus, swine vesicular disease virus, and vesicular exanthema of swine virus. To estimate diagnostic sensitivity, 159 epithelial samples collected between 1996 and 2002 from naturally infected cattle in Colombia were used. The assay cut off was calculated by testing RNA extracted from 150 virus-negative bovine tissues consisting of tongue, soft palate, muzzle, coronary band, and lymph node. All infected cattle were test positive for VS by results of real-time RT-PCR analysis; results for 156 of 159 (98.1%) agreed with the serotype determination from the complement-fixation test. Amplification did not occur in any of the negative bovine epithelial samples, allowing the cut-off values for the assay to be set. The real-time RT-PCR assay was documented to be sensitive and specific for the detection of VSV-NJ and VSV-IN (1-3) strains from field samples in a single reaction, thereby supporting use of this assay in the differential diagnosis of vesicular virus diseases in cattle.

Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer

Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous detection and differentiation of three Office International des Epizooties (OIE) classified vesicular viruses: foot-and-mouth disease virus, vesicular stomatitis virus and swine vesicular disease, causing clinically indistinguishable vesicular diseases in swine. The multiplex assay consists of extraction of total RNA from clinical samples; reverse transcription to cDNA using random primers and one-tube real-time amplification of cDNA using multiplex PriProET with specific fluorescent-labelled primers and probes for detection of the three viruses from the vesicular disease complex. The probes are labelled with unique reporter fluorophores, which during amplification are excited by donor fluorophores incorporated in the 5′ end of specific amplicons by primer extension. The sensitivity of the multiplex assay was approximately 100 TCID 50, which is 10-fold lower compared to the individual PriProET assays for the three vesicular viruses.

Sensitivity of primary cells immortalised by oncogene transfection for the detection and isolation of foot-and-mouth disease and swine vesicular disease viruses

Primary cells derived from calf thyroid (CTY), calf kidney (CK) and piglet kidney (PK) were immortalised by oncogene transfection and their susceptibility to infection by foot-and-mouth disease (FMD) virus and swine vesicular disease (SVD) virus examined. Eighty-five immortalised cell lines (47 CTY, 20 CK and 18 PK) proved stable upon repeated cell culture passage and many supported the growth of FMD virus and several of the PK cell lines supported SVD virus. However, none of the immortalised lines exhibited either the degree of sensitivity or the specificity for all virus serotypes and strains as shown by primary CTY and IB-RS-2 cell cultures which are routinely employed for vesicular virus diagnosis.

The World Reference Laboratory for Foot and Mouth Disease: a review of thirty-three years of activity (1958-1991).

The range of activities and contributions of the World Reference Laboratory for Foot and Mouth Disease in Pirbright, Surrey, United Kingdom, from 1958 to 1991 is reviewed. The countries for which a service has been provided, the number of samples submitted for investigation and the serotypes identified are recorded. Factors which have influenced the number of samples received are outlined. The developments and improvements made in the laboratory diagnosis of vesicular virus diseases over the thirty-three-year period are described.

Selection of foot and mouth disease antisera for diagnosis by ELISA.

The World Reference Laboratory (WRL) for foot and mouth disease receives vesicular virus samples from throughout the world for the diagnosis and serotype identification of foot and mouth disease virus (FMDV). These samples may contain strains of virus which are widely different antigenically. To optimise the efficiency of laboratory diagnosis, it is essential that the antisera used in diagnostic procedures be specific, of high titre and broadly reactive within that serotype in order to detect all strains of virus currently circulating in the field. Experience and investigation by complement fixation have shown that antisera against certain strains of FMDV fulfil this requirement better than others (3). Many of these reagents had been produced from the hyperimmunisation of guinea pigs (1). The preferred method for the production of reference antisera now involves the utilisation of inactivated and purified FMDV antigens. In addition, over the last two years an enzyme-linked immunosorbent assay (ELISA; 9) has been extensively evaluated and adopted for routine use (6). Rabbit and guinea pig antisera to inactivated 146S antigens have consequently been produced from the current (1985) diagnostic strains and those suggested as alternatives (3). These reagents have been assessed in the ELISA to extend previous studies for the selection of sera for diagnosis.

Plaque production by carrier strains of foot-and-mouth disease virus in BHK-monolayers incubated at different temperatures.

Type C vesicular and carrier virus isolates from two farms were compared with regard to plaque formation in BHK-monolayers incubated at different temperatures. At temperatures below 37°C differences between the two categories were only slight. When incubated at higher temperatures, especially at 41°C late carrier isolates produced a smaller number of plaques than early ones or vesicular virus. Plaques formed by vesicular isolates reached maximal diameters when incubated at 40°C, whereas all carrier viruses developed the largest plaques at temperatures of 37°C.

Detection of complement fixing antibodies against foot-and-mouth disease in cattle serum.

The complement fixation test has become of considerable value in the early diagnosis of foot-and-mouth disease (F and M) in cattle and in typing the virus from this disease. However, this method is less useful in later stages of the disease when infected tissue can no longer be obtained for antigen. Furthermore, it is difficult to detect complement fixing antibodies in serums from convalescent cattle when using conventional procedures. Therefore, many attempts have been made to improve the sensivitity of the test. These modifications of the complement fixation test have included the use of unheated serum (Fredericks, 1949) or performance of the entire test at higher salt concentration (Serra and Guarini, 1951). None of these have proved to be successful. An indirect method for demonstrating antibodies in late convalescent F and M serums has been suggested by Rice and Brooksby (1953). The indirect complement fixation test had earlier been applied by Rice (1948) in detecting antibodies in anti-pullorum serums, by Wolfe (1949) in anti-Newcastle disease serums, by Karrer et al (1950) and Hilleman et al (1951) in anti-ornithosispsittacosis, and by Downie et al (1950) in anti-variola serums from chickens. Rice and Boulanger (1953) have also used it to demonstrate antibodies against vesicular viruses in cattle, and Rice and McKercher (1954) in studies of vesicular stomatitis in horses, cattle and swine. The difficulty with the indirect test is its relative complexity, and it is time consuming. A simple modification of the F and M complement fixation test was described by Nordberg and Schjerning-Thiesen (1953) in a preliminary report. These workers were able to demonstrate antibodies against F and M in the serums of three hyperimmunized and seven convalescent cattle when a conventional test was employed but with temporary hypertonization of the serum component alone. The present report describes studies on a larger series of convalescent and vaccinated cows and presents the procedure in greater detail.