Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the family Bunyaviridae, genus Nairovirus. The virus is transmitted to humans through infected tick bites or from direct contact with viremic animals or humans. In the present study, a total of 1,015 adult ticks were collected from cattle (603 specimens), sheep (17 specimens), and goats (395 specimens) in the Kelkit Valley in Turkey. Four tick species were recognized on the animals in the surveyed region. The most abundant species were Rhipicephalus bursa and Hyalomma marginatum marginatum, at 47.68% (484/1,015) and 46.40% (471/1,015), respectively. Reverse transcriptase PCR was used to recover partial sequences of the CCHFV small (S) genome segment. The presence of CCHFV was determined in 3 of 33 (9.09%) R. bursa pools and in 1 of 31 (3.22%) H. m. marginatum pools. Virus sequences from R. bursa were extremely different from those of the Greek CCHFV strain (U04958) isolated from an R. bursa tick. Phylogenetic analysis indicated that the CCHFV isolates obtained in this study clustered in group 5, whose range encompasses southwestern Russian and Kosovo. This is the first evidence of CCHFV in ticks from Turkey. Even though Hyalomma is the main vector for CCHFV, R. bursa may play a role in CCHFV transmission.

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.

Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5 and 3 coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (GGUAGAC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (؊17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5 end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.Coronaviruses and arteriviruses, both members of the Nidovirus order of plus-strand RNA animal viruses, appear unique among RNA viruses in their use of a discontinuous transcription step during synthesis of subgenomic mRNAs (10,14,28,48,54). In both groups of viruses, the transcription pathway ultimately yields a 3Ј coterminal nested set of subgenomic mRNAs that are also 5Ј coterminal with the virus genome. The common 5Ј-terminal sequence, called the "leader," encoded at the genome 5Ј terminus, makes up only a portion of the 5Ј untranslated region in the genome and in each subgenomic mRNA (sgmRNA) species. In general, translation occurs most abundantly from the 5Ј-most open reading frame (ORF) on each sgmRNA. When originally described, the leader was postulated to become fused with the sgmRNA species by a leader-priming mechanism wherein the RdRp undergoes a copy choice jump on the virus genome-length minus-strand template during plus-strand synthesis (4). The jump in this model would occur for each sgmRNA molecule synthesized, and a postulated 3Ј35Ј exonuclease would trim the large primer (80 to 140 nucleotides [nt]), termed free leader, d...

Coronaviruses are positive-strand, RNA-dependent RNA polymerase-utilizing viruses that require a polymerase template switch, characterized as discontinuous transcription, to place a 5-terminal genomic leader onto subgenomic mRNAs (sgmRNAs). The usually precise switch is thought to occur during the synthesis of negative-strand templates for sgmRNA production and to be directed by heptameric core donor sequences within the genome that match an acceptor core (UCUAAAC in the case of bovine coronavirus) near the 3 end of the 5-terminal genomic leader. Here it is shown that a 22-nucleotide (nt) donor sequence engineered into a packageable bovine coronavirus defective interfering (DI) RNA and made to match a sequence within the 65-nt virus genomic leader caused a template switch yielding an sgmRNA with only a 33-nt minileader. By changing the donor sequence, acceptor sites between genomic nt 33 and 97 (identical between the DI RNA and the viral genome) could be used to generate sgmRNAs detectable by Northern analysis (ϳ2 to 32 molecules per cell) by 24 h postinfection. Whether the switch was intramolecular only was not determined since a potentially distinguishing acceptor region in the DI RNA rapidly conformed to that in the helper virus genome through a previously described template switch known as leader switching. These results show that crossover acceptor sites for discontinuous transcription (i) need not include the UCUAAAC core and (ii) rest within a surprisingly wide 5-proximal "hotspot." Overlap of this hotspot with that for leader switching and with elements required for RNA replication suggests that it is part of a larger 5-proximal multifunctional structure.Among the known positive-strand RNA viruses that use an RNA-dependent RNA polymerase (RdRp) for replication, only members of the Coronaviridae (11) and Arteriviridae (45) families in the order Nidovirales (12) require a 3Ј-coterminal nested set of subgenomic mRNAs (sgmRNAs) on which a common leader sequence, encoded only at the 5Ј end of the genome, is placed. Curiously, not all families in the Nidovirales order have the same requirement. In the Toroviridae, for example, only one of the three sgmRNA species possesses a leader in common with the genome (51), and in the Roniviridae, no common leader on sgmRNAs is found (9). Thus, precisely how nidoviruses differ with regard to the mechanisms of sgmRNA synthesis remains to be determined. Attachment of the leader to sgmRNAs involves a mechanism of high-frequency RdRp template switching (i.e., discontinuous transcription) that is not fully understood but may be a target for designed molecular intervention against such infections as those caused by the severe acute respiratory syndrome coronavirus (17).Results from several recent studies have generally supported a model for coronavirus discontinuous transcription (34, 36) in which positive-strand-to-positive-strand RdRp template switching occurs during the generation of sgmRNA-length negativestrand templates (14,16,41,42) that are then used reiteratively in do...

Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne zoonotic disease. The disease has been reported in many countries of Africa, Asia, the Middle East, and in Eurasia. During the past decade, new foci of CCHF have emerged in the Balkan Peninsula, southwest Russia, the Middle East, western China, India, Africa, and Turkey. CCHF virus produces severe hemorrhagic manifestations in humans with fatality rates up to 30%. Vaccine development efforts have been significantly hampered by a lack of animal models and therefore, no protective vaccine has been achieved. Lately, IFN α/β receptor deficient (IFNAR−/−) mice have been established as a novel small animal model of CCHF virus infection. In the present study, we found that IFNAR−/− mice highly susceptible to CCHF virus Turkey-Kelkit06 strain. Immunization with the cell culture based vaccine elicited a significant level of protection against high dose challenge (1,000 PPFU) with a homologous CCHF virus in IFNAR−/− mice.