Critical parameters of real time reverse transcription polymerase chain reaction (RT-PCR) diagnostics: Sensitivity and specificity for bluetongue virus.

A new variant of bluetongue virus serotype 3, BTV3 ITL 2018 (here named: BTV3), was included in serial dilutions in the BT Proficiency Test 2020. Although the OIE-recommended panBTV real time RT-PCR test targeting genome segment 10 (Seg-10) detected this variant, we showed that reverse transcription (RT) at 61 °C instead of 50 °C completely abolished detection. Another Seg-10 panBTV real time RT-PCR test detected BTV3, irrespective of the temperature of RT. In silico validation showed that each of the OIE-recommended PCR primers using IVI-primers contain single mismatches at the -3 position for BTV3. In contrast, WBVR-primers of a second test completely match to the BTV3 variant. Our results suggest that single mismatches caused false negative PCR results for BTV3 at high RT temperature. Indeed, correction of both IVI-primers for BTV3 led to positive results for BTV3 but negative results for all other samples of the BT Proficiency Test 2020. Apparently, variability of the -3 position is sufficient for discriminative PCR detection, although the single mismatch in the IVI-reverse primer was the most important for this phenomenon. Extensive in silico validation showed that targets of both Seg-10 panBTV RT-PCR tests are not completely conserved, and the detailed effect of single mismatches are hard to predict. Therefore, we recommend at least two panBTV RT-PCR tests to minimize the risk of false negatives. Preferably, their PCR targets should be located at completely different and highly conserved regions of the BTV genome to guarantee adequate detection of future BTV infections.

Two novel porcine teschovirus strains as the causative agents of encephalomyelitis in the Netherlands.

BACKGROUND: Porcine teschovirus (PTV) circulates among wild and domesticated pig populations without causing clinical disease, however neuroinvasive strains have caused high morbidity and mortality in the past. In recent years, several reports appeared with viral agents as a cause for neurologic signs in weanling and growing pigs among which PTV and new strains of PTV were described. CASE PRESENTATION: On two unrelated pig farms in the Netherlands the weanling pig population showed a staggering gate, which developed progressively to paresis or paralysis of the hind legs with a morbidity up to 5%. After necropsy we diagnosed a non-suppurative encephalomyelitis on both farms, which was most consistent with a viral infection. PTV was detected within the central nervous system by qPCR. From both farms PTV full-length genomes were sequenced, which clustered closely with PTV-3 (98%) or PTV-11 (85%). Other common swine viruses were excluded by qPCR and sequencing of the virus. CONCLUSION: Our results show that new neuroinvasive PTV strains still emerge in pigs in the Netherlands. Further research is needed to investigate the impact of PTV and other viral agents causing encephalomyelitis within wild and domestic pig populations supported by the awareness of veterinarians.

Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus.

BACKGROUND: Transmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1-24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25-27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called 'differential virus replication' in vitro. METHODS: Virus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. RESULTS: A 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into 'corrections' of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo. CONCLUSION: Small changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.

Diagnostic DIVA tests accompanying the Disabled Infectious Single Animal (DISA) vaccine platform for African horse sickness.

African Horse Sickness Virus (AHSV) (Orbivirus genus, Reoviridae family) causes high mortality in naïve domestic horses with enormous economic and socio-emotional impact. There are nine AHSV serotypes showing limited cross neutralization. AHSV is transmitted by competent species of Culicoides biting midges. AHS is a serious threat beyond the African continent as endemic Culicoides species in moderate climates transmit the closely related prototype bluetongue virus. There is a desperate need for safe and efficacious vaccines, while DIVA (Differentiating Infected from Vaccinated) vaccines would accelerate control of AHS. Previously, we have shown that highly virulent AHSV with an in-frame deletion of 77 amino acids (aa) in NS3/NS3a is completely safe, does not cause viremia and shows protective capacity. This deletion mutant is a promising DISA (Disabled Infectious Single Animal) vaccine platform, since exchange of serotype specific virus proteins has been shown for all nine serotypes. Here, we show that a prototype NS3 competitive ELISA is DIVA compliant to AHS DISA vaccine platforms. Epitope mapping of NS3/NS3a shows that more research is needed to evaluate this prototype serological DIVA assay regarding sensitivity and specificity, in particular for AHSVs expressing antigenically different NS3/NS3a proteins. Further, an experimental panAHSV PCR test targeting genome segment 10 is developed that detects reference AHSV strains, whereas AHS DISA vaccine platforms were not detected. This DIVA PCR test completely guarantees genetic DIVA based on in silico and in vitro validation, although test validation regarding diagnostic sensitivity and specificity has not been performed yet. In conclusion, the prototype NS3 cELISA and the PCR test described here enable serological and genetic DIVA accompanying AHS DISA vaccine platforms.

Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis.

BACKGROUND: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines. METHODS: The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized Culicoides sonorensis midges was investigated. Virus strains were generated using reverse genetics and their growth was examined in vitro. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. RESULTS: Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides. CONCLUSION: The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges.

Sustained high-throughput polymerase chain reaction diagnostics during the European epidemic of Bluetongue virus serotype 8.

A real-time reverse transcription polymerase chain reaction assay (PCR test) based on genome segment 10 of Bluetongue virus (BTV) was developed. The PCR test consists of robotized viral RNA isolation from blood samples and an all-in-one method including initial denaturation of genomic double-stranded RNA, reverse transcription polymerase chain reaction (RT-PCR), and real-time detection and analysis. Reference strains of the 24 recognized BTV serotypes, isolates from different years, and geographic origins were detected. Other orbiviruses such as African horse sickness virus, Epizootic hemorrhagic disease virus, and Equine encephalosis virus were not detected. Experimentally infected animals were PCR positive from 2 days postinoculation, which was earlier than fever, other clinical signs, or seroconversion. The diagnostic sensitivity and specificity were very close to or even 100%. The PCR test played a key role in the detection of BTV serotype 8 in August 2006 in The Netherlands. The outbreak in a completely naive ruminant population allowed for further evaluation of the PCR test with field samples. In 2006, the correlation between enzyme-linked immunosorbent assay and PCR results was estimated to be 95%. In the following years, the PCR test was used for diagnosis of diseased animals, for testing of healthy animals for trade purposes, and for detection of BTV RNA in different species of the insect vector, Culicoides. In the autumn of 2008, BTV serotype 6 unexpectedly emerged in northwest Europe and was also detected with the PCR test developed in the current study. The performance in routine use over 5 years has been recorded and evaluated.

Use of a codified medication process for documentation of home medications.

To determine the quality and completeness of the list of home medications documented by nurses using a codified process, authors conducted a comparative study of home medications using a non-codified and codified process for documentation of required data fields including drug, dose, route of administration, frequency, and schedule. Each documented home medication (DHM) was evaluated based on the ability to convert to an inpatient medication order. The home medication was classified as non-convertible if one or more of the required data fields were missing, inaccurate, or incomplete. The study compared 176 patients with 1618 DHM in the non-codified group to 94 patients with 646 DHM in the codified group. All DHM could be converted to inpatient orders for 70% of the patients in the codified group compared with 42% in the non-codified group. Based on each DHM, the codified process resulted in 92% of the DHM being able to convert to inpatient orders compared with 82% for the non-codified process. Authors conclude that use of a codified process to document home medications has the potential to increase the number of complete drug entries and in the number of patients with a DHM list in which all of the medication entries have all of the dosing information.

Potential role of ticks as vectors of bluetongue virus.

When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not to survive the northern European winter, and transovarial transmission in Culicoides is not recorded, we examined the potential vector role of ixodid and argasid ticks for bluetongue virus. Four species of ixodid ticks (Ixodes ricinus, Ixodes hexagonus, Dermacentor reticulatus and Rhipicephalus bursa) and one soft tick species, Ornithodoros savignyi, ingested BTV8-containing blood either through capillary feeding or by feeding on artificial membranes. The virus was taken up by the ticks and was found to pass through the gut barrier and spread via the haemolymph into the salivary glands, ovaries and testes, as demonstrated by real-time reverse transcriptase PCR (PCR-test). BTV8 was detected in various tissues of ixodid ticks for up to 21 days post feeding and in Ornithodoros ticks for up to 26 days. It was found after moulting in adult Ixodes hexagonus and was also able to pass through the ovaries into the eggs of an Ornithodoros savignyi tick. This study demonstrates that ticks can become infected with bluetongue virus serotype 8. The transstadial passage in hard ticks and transovarial passage in soft ticks suggest that ticks have potential vectorial capacity for bluetongue virus. Further studies are required to investigate transmission from infected ticks to domestic livestock. This route of transmission could provide an additional clue in the unresolved mystery of the epidemiology of Bluetongue in Europe by considering ticks as a potential overwintering mechanism for bluetongue virus.

Validation of a LightCycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus.

A specific reverse transcription polymerase chain reaction (RT-PCR) for the detection of the polymerase gene (3D) of foot-and-mouth disease virus (FMDV) was developed and validated with an analytical sensitivity of equal to, to 1,000 times higher than that of a single passage virus isolation. The performance of the RT-PCR was determined in 180 runs. After implementation, 5.3% of the tests had to be rejected due to invalid controls (e.g. cross-contamination of negative controls). The diagnostic sensitivity, determined using 124 samples from experimentally infected animals, was 91.9% for RT-PCR and 84.7% for virus isolation. Diagnostic specificity, determined by testing 258 samples from uninfected animals, was 100% by both tests. Of the 627 samples tested by RT-PCR and virus isolation, 85 reacted positively in both tests (13.5%) and 447 negatively in both tests (71.3%). One sample was positive by virus isolation and negative by RT-PCR (0.2%), 94 samples were positive by RT-PCR and negative by virus isolation (15%). The majority (84 of 94) of the 15% RT-PCR positive and virus isolation negative samples were among other samples from farms that reacted positively by both tests. The new RT-PCR is a robust, reliable and sensitive test, provided that adequate measures are taken to prevent cross-contamination. A possible preventive measure is to exclude ELISA positive samples from the RT-PCR testing.