Co-Circulation of West Nile, Usutu, and Tick-Borne Encephalitis Viruses in the Same Area: A Great Challenge for Diagnostic and Blood and Organ Safety.

Viral infections caused by viruses from the family Flaviviridae such as Zika (ZIKV), Dengue (DENV), yellow fever (YFV), tick-borne encephalitis (TBEV), West Nile (WNV), and Usutu (USUV) are some of the most challenging diseases for recognition in clinical diagnostics and epidemiological tracking thanks to their short viremia, non-specific symptoms, and high cross-reactivity observed in laboratory techniques. In Central Europe, the most relevant endemic flaviviruses are mosquito-borne WNV and USUV, and tick-borne TBEV. All three viruses have been recognised to be responsible for human neuroinvasive diseases. Moreover, they are interrupting the blood and transplantation safety processes, when the great efforts made to save a patient's life could be defeated by acquired infection from donors. Due to the trend of changing distribution and abundance of flaviviruses and their vectors influenced by global change, the co-circulation of WNV, USUV, and TBEV can be observed in the same area. In this perspective, we discuss the problems of flavivirus diagnostics and epidemiology monitoring in Slovakia as a model area of Central Europe, where co-circulation of WNV, USUV, and TBEV in the same zone has been recently detected. This new situation presents multiple challenges not only for diagnostics or surveillance but particularly also for blood and organ safety. We conclude that the current routinely used laboratory diagnostics and donor screening applied by the European Union (EU) regulations are out of date and the novel methods which have become available in recent years, e.g., next-gene sequencing or urine screening should be implemented immediately.

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B.

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.

Surveillance of SARS-CoV-2 lineage B.1.1.7 in Slovakia using a novel, multiplexed RT-qPCR assay.

The emergence of a novel SARS-CoV-2 B.1.1.7 variant sparked global alarm due to increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack genomic surveillance programs and failed assays detect unrelated variants containing similar mutations as B.1.1.7, we used allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop an RT-qPCR test that accurately and rapidly differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing and conducted a country-wide surveillance study of B.1.1.7 prevalence in Slovakia. Our multiplexed RT-qPCR test showed 97% clinical sensitivity and retesting 6,886 SARS-CoV-2 positive samples obtained during three campaigns performed within one month, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.

Genetic Characterization of a Neurovirulent West Nile Virus Variant Associated with a Fatal Great Grey Owl Infection.

This study reports on a fatal case of a captive great grey owl infected with the West Nile virus (WNV) in the zoological garden Kosice, eastern Slovakia (Central Europe). The tissue samples of the dead owl were used for virus isolation and genetic characterization. The novel isolate is genetically closer to Hungarian, Greek, and Bulgarian strains from the central/southern European clade of lineage 2 than to the strains previously isolated in Slovakia. Interestingly, it carries NS3-249P, a molecular virulence determinant associated with higher neurovirulence, which has not previously been observed in Slovakia. Subsequent serological investigation of the captive owls revealed additional seropositive animals, indicating local WNV transmission. Although no WNV-positive mosquitoes were found, the presence of the WNV principal vector Culex pipiens complex together with the described fatal case and further serological findings indicate an endemic focus of bird-neurovirulent WNV variant in the area.

Alimentary Infections by Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) causes serious the neurological disease, tick-borne encephalitis (TBE). TBEV can be transmitted to humans by ticks as well as by the alimentary route, which is mediated through the consumption of raw milk products from infected ruminants such as sheep, goats, and cows. The alimentary route of TBEV was recognized in the early 1950s and many important experimental studies were performed shortly thereafter. Nowadays, alimentary TBEV infections are recognized as a relevant factor contributing to the overall increase in TBE incidences in Europe. This review aims to summarize the history and current extent of alimentary TBEV infections across Europe, to analyze experimental data on virus secretion in milk, and to review possible alimentary infection preventive measures.

Dermacentor reticulatus is a vector of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV; family Flaviviridae) is the most medically important tick-borne virus in Europe and Asia. Ixodes ricinus and I. persulcatus ticks are considered to be the main vector ticks of TBEV in nature due to their specific ecological associations with the vertebrate hosts. Nevertheless, recent TBEV prevalence studies in ticks suggest that Dermacentor reticulatus ticks might play a relevant role in the maintenance of TBEV in nature. The goal of this study was to evaluate the vector competency of D. reticulatus for TBEV through experimental tick infections and comparative in vivo transmission studies involving D. reticulatus and I. ricinus ticks. We observed that after a transcoxal micro-capillary inoculation, adult female D. reticulatus ticks efficiently replicated TBEV during the observed period of 21 days. The mean virus load reached up to 2.5 × 105 gene copies and 6.4 × 104 plaque forming units per tick. The infected D. reticulatus ticks were able to transmit the virus to mice. The course of infection in mice was comparable to the infection after a tick bite by I. ricinus while the virus spread and clearance was slightly faster. Moreover, D. reticulatus ticks were capable of tick-to-tick non-viraemic transmission of TBEV to the Haemaphysalis inermis nymphs during co-feeding on the same animal. The co-feeding transmission efficiency was overall slightly lower (up to 54 %) in comparison with I. ricinus (up to 94 %) and peaked 1 day later, at day 3. In conclusion, our study demonstrated that D. reticulatus is a biologically effective vector of TBEV. In line with the recent reports of its high TBEV prevalence in nature, our data indicate that in some endemic foci, D. reticulatus might be an underrecognized TBEV vector which contributes to the expansion of the TBEV endemic areas.

Counterattacking the tick bite: towards a rational design of anti-tick vaccines targeting pathogen transmission.

Hematophagous arthropods are responsible for the transmission of a variety of pathogens that cause disease in humans and animals. Ticks of the Ixodes ricinus complex are vectors for some of the most frequently occurring human tick-borne diseases, particularly Lyme borreliosis and tick-borne encephalitis virus (TBEV). The search for vaccines against these diseases is ongoing. Efforts during the last few decades have primarily focused on understanding the biology of the transmitted viruses, bacteria and protozoans, with the goal of identifying targets for intervention. Successful vaccines have been developed against TBEV and Lyme borreliosis, although the latter is no longer available for humans. More recently, the focus of intervention has shifted back to where it was initially being studied which is the vector. State of the art technologies are being used for the identification of potential vaccine candidates for anti-tick vaccines that could be used either in humans or animals. The study of the interrelationship between ticks and the pathogens they transmit, including mechanisms of acquisition, persistence and transmission have come to the fore, as this knowledge may lead to the identification of critical elements of the pathogens' life-cycle that could be targeted by vaccines. Here, we review the status of our current knowledge on the triangular relationships between ticks, the pathogens they carry and the mammalian hosts, as well as methods that are being used to identify anti-tick vaccine candidates that can prevent the transmission of tick-borne pathogens.

Ultracentrifugation enrichment protocol followed by total RNA sequencing allows assembly of the complete mitochondrial genome.

The mitochondrial genome is an independent genetic system in each eukaryotic cell outside the nuclear genome. Mitochondrial DNA (mtDNA) appears in high copy number within one cell, unlike nuclear DNA, which exists in two copies. But nevertheless, mtDNA represent only small part of total cellular DNA what causes problematic analysis and identification of relevant mutations. While most researchers tend to overlook it because of its small size, the mitochondrial genome contains genes that are essential for cellular energetics and survival. Because of the increased awareness on the importance of metabolism and bioenergetics in a wide variety of human diseases, more and more mtDNA studies were performed. Mitochondrial genome research has established the connection between mtDNA and a wide variety of diseases such as cancer or neurodegenerative disorders. At the present time, several methods are known, that allow sequencing of mtDNA. However, genomic analysis is often complicated due to the low content of mtDNA compared to nuclear DNA. For this reason, we have designed a new approach to obtaining the genomic mitochondrial sequence. We chose RNA based sequencing. Since human mtDNA does not contain introns, the reconstruction of whole mitochondrial genome through RNA sequencing seems to be effective. Our method is based on total RNA sequencing coupled with simple ultracentrifugation protocol and de novo assembly. Following our protocol, we were able to assemble a complete mammalian mitochondrial genome with a length of 16,505 bp and an average coverage of 156. The method is a relatively simple and inexpensive which could help in the further research or diagnostics of mtDNA-based diseases.

A Novel Hantavirus of the European Mole, Bruges Virus, Is Involved in Frequent Nova Virus Coinfections.

Hantaviruses are zoonotic viruses with a complex evolutionary history of virus-host coevolution and cross-species transmission. Although hantaviruses have a broad reservoir host range, virus-host relationships were previously thought to be strict, with a single virus species infecting a single host species. Here, we describe Bruges virus, a novel hantavirus harbored by the European mole (Talpa europaea), which is the well-known host of Nova virus. Phylogenetic analyses of all three genomic segments showed tree topology inconsistencies, suggesting that Bruges virus has emerged from cross-species transmission and ancient reassortment events. A high number of coinfections with Bruges and Nova viruses was detected, but no evidence was found for reassortment between these two hantaviruses. These findings highlight the complexity of hantavirus evolution and the importance of further investigation of hantavirus-reservoir relationships.

Phylogenetic analysis of Puumala virus strains from Central Europe highlights the need for a full-genome perspective on hantavirus evolution.

Puumala virus (PUUV), carried by bank voles (Myodes glareolus), is the medically most important hantavirus in Central and Western Europe. In this study, a total of 523 bank voles (408 from Germany, 72 from Slovakia, and 43 from Czech Republic) collected between the years 2007-2012 were analyzed for the presence of hantavirus RNA. Partial PUUV genome segment sequences were obtained from 51 voles. Phylogenetic analyses of all three genome segments showed that the newfound strains cluster with other Central and Western European PUUV strains. The new sequences from Sumava (Bohemian Forest), Czech Republic, are most closely related to the strains from the neighboring Bavarian Forest, a known hantavirus disease outbreak region. Interestingly, the Slovak strains clustered with the sequences from Bohemian and Bavarian Forests only in the M but not S segment analyses. This well-supported topological incongruence suggests a segment reassortment event or, as we analyzed only partial sequences, homologous recombination. Our data highlight the necessity of sequencing all three hantavirus genome segments and of a broader bank vole screening not only in recognized endemic foci but also in regions with no reported human hantavirus disease cases.

Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

Phylogenetic analysis of a newfound bat-borne hantavirus supports a laurasiatherian host association for ancestral mammalian hantaviruses.

Until recently, hantaviruses (family Bunyaviridae) were believed to originate from rodent reservoirs. However, genetically distinct hantaviruses were lately found in shrews and moles, as well as in bats from Africa and Asia. Bats (order Chiroptera) are considered important reservoir hosts for emerging human pathogens. Here, we report on the identification of a novel hantavirus, provisionally named Makokou virus (MAKV), in Noack's Roundleaf Bat (Hipposideros ruber) in Gabon, Central Africa. Phylogenetic analysis of the genomic l-segment showed that MAKV was the most closely related to other bat-borne hantaviruses and shared a most recent common ancestor with the Asian hantaviruses Xuan Son and Laibin. Breakdown of the virus load in a bat animal showed that MAKV resembles rodent-borne hantaviruses in its organ distribution in that it predominantly occurred in the spleen and kidney; this provides a first insight into the infection pattern of bat-borne hantaviruses. Ancestral state reconstruction based on a tree of l gene sequences of all relevant hantavirus lineages was combined with phylogenetic fossil host hypothesis testing, leading to a statistically significant rejection of the mammalian superorder Euarchontoglires (including rodents) but not the superorder Laurasiatheria (including shrews, moles, and bats) as potential hosts of ancestral hantaviruses at most basal tree nodes. Our data supports the emerging concept of bats as previously overlooked hantavirus reservoir hosts.

Immunization with recombinant subolesin does not reduce tick infection with tick-borne encephalitis virus nor protect mice against disease.

Tick-borne encephalitis (TBE) is a growing zoonotic disease caused by tick-borne encephalitis virus (TBEV) infection. Although effective vaccines for TBEV are available, on-going vaccination efforts are insufficient to prevent increase in TBE cases annually. Vaccination with arthropod vector antigens to reduce vector infestations and vector capacity allows control of several vector-borne diseases by targeting their common vector. Subolesin (SUB) is a tick protective antigen that has a role in tick innate immunity and other molecular pathways and has been shown to protect against tick infestations and infection by vector-borne pathogens. However, SUB expression and the effect of SUB immunization have not been evaluated for tick-borne viruses. Herein, we showed that SUB expression is downregulated during Ixodes ricinus tick feeding but induced in ticks infected with TBEV, thus supporting a role for this molecule in tick innate immune response to virus infection. Immunization with recombinant SUB reduced SUB mRNA levels in nymphs co-feeding with infected females and suggested and effect on tick infestations in mice. However, SUB immunization did not reduce tick infection with TBEV nor protect mice against TBE. These results suggested that SUB is not a good candidate antigen for vaccination against TBEV and support the characterization of tick-pathogen interactions to identify mechanisms that could be targeted to reduce TBEV infection and transmission by ticks.

An antivector vaccine protects against a lethal vector-borne pathogen.

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a "dual action" in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: "transmission," number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; "support," number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and "survival," number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.

Tick-borne encephalitis virus foci in Slovakia.

Tick-borne encephalitis (TBE) virus as a typical arbovirus relies on two types of hosts for its survival: ticks act both as virus vectors and reservoir hosts, and vertebrates amplify the virus infection by acting as a source of infection for feeding ticks. Longitudinal monitoring of TBE virus in ticks and vertebrate hosts including humans over a period of 40 years resulted in the identification of the areas of Slovakia where TBE virus is endemic. These are concentrated to the western, southern, and eastern parts of the country. Even with recently identified foci there is no evidence that the size and location of the natural TBE foci have changed significantly during the last decades. Numbers of diagnosed hospitalised cases of TBE in Slovakia vary from less than 20 to almost 100 cases annually with 54-89 cases in recent years. A part of these cases (33 cases during the last 5 years) are alimentary infections after drinking of raw goat and sheep milk.