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Background Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike protein to bind to a host cell surface receptor. The ongoing outbreak of SARS-CoV-2 inculcates the need for the development of effective intervention strategies. Objectives In this study, we aim to produce pseudotyped coronaviruses of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 and show its applications, including virus entry, neutralization, and screening of entry inhibitors from natural products. Methods Here, we generated VSV-based pseudotyped coronaviruses (CoV-PVs) for SARS-CoV-1, MERS-CoV, and SARS-CoV-2. Recombinant spike proteins of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 were transiently expressed in HEK293T cells followed by infection with recombinant VSV. High titer pseudoviruses were harvested and subjected to distinct validation assays, which confirms the proper spike pseudotyping. Further, specific receptor-mediated entry was confirmed by antibody neutralization and soluble form of receptor inhibition assay on Vero E6 cells. Next, these CoV-PVs were used for screening of antiviral activity of natural compounds such as green tea and Spirulina extract. Results Medicinal plants and natural compounds have been traditionally used as antiviral agents. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind to their receptors for cellular entry. SARS-CoV-1 and MERS-CoV anti-sera neutralize SARS-CoV-1-PV and SARS-CoV-2-PV, and MERS-CoV-PV, respectively. Incubation of soluble ACE2 with CoV-PVs inhibited entry of SARS-CoV-1 and SARS-CoV-2 PVs but not MERS-CoV-PV. Also, transient expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARS-CoV-1-PV, SARS-CoV-2-PV, and MERS-CoV-PV, respectively. Next, we showed the antiviral properties of known entry inhibitors of enveloped viruses, Spirulina, and green tea extracts against CoV-PVs. SARS-CoV-1-PV, MERS-CoV-PV, and SARS-CoV-2-PV entry was blocked with higher efficiency when preincubated with either green tea or Spirulina extracts. Green tea provided a better inhibitory effect by binding to the S1 domain of the spike and blocking the spike interaction with its receptor. Conclusion In summary, we demonstrated that pseudotyped viruses are an ideal tool for studying viral entry, quantification of neutralizing antibodies, and screening of entry inhibitors in a BSL-2 facility. Moreover, green tea might be a promising natural remedy against emerging coronaviruses.

Recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 initiate virus infection by binding of their spike glycoprotein with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and enter into the host cells mainly via the clathrin-mediated endocytosis pathway. However, the internalization process post attachment with the receptor is not clear for both SARS-CoV-1 and -2. Understanding the cellular factor/s or pathways used by these CoVs for internalization might provide insights into viral pathogenesis, transmission, and development of novel therapeutics. Here, we demonstrated that the cytoplasmic tail of ACE2 is not essential for the entry of SARS-CoV-1 and -2 by using bioinformatics, mutational, confocal imaging, and pseudotyped SARS-CoVs infection studies. ACE2 cytoplasmic domain (cytACE2) contains a conserved internalization motif and eight putative phosphorylation sites. Complete cytoplasmic domain deleted ACE2 (∆cytACE2) was properly synthesized and presented on the surface of HEK293T and BHK21 cells like wtACE2. The SARS-CoVs S1 or RBD of spike protein binds and colocalizes with the receptors followed by internalization into the host cells. Moreover, pseudotyped SARS-CoVs entered into wtACE2- and ∆cytACE2-transfected cells but not into dipeptidyl peptidase 4 (DPP4)-expressing cells. Their entry was significantly inhibited by treatment with dynasore, a dynamin inhibitor, and NH4Cl, an endosomal acidification inhibitor. Furthermore, SARS-CoV antibodies and the soluble form of ACE2-treated pseudotyped SARS-CoVs were unable to enter the wtACE2 and ∆cytACE2-expressing cells. Altogether, our data show that ACE2 cytoplasmic domain signaling is not essential for the entry of SARS-CoV-1 and -2 and that SARS-CoVs entry might be mediated via known/unknown host factor/s.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Ferret coronaviruses (FRCoVs) exist as an enteric and a systemic pathotype, of which the latter is highly lethal to ferrets. To our knowledge, this study provides the first full genome sequence of a FRCoV, tentatively called FRCoV-NL-2010, which was detected in 2010 in ferrets in The Netherlands. Phylogenetic analysis showed that FRCoV-NL-2010 is most closely related to mink CoV, forming a separate clade of mustelid alphacoronavirus that split off early from other alphacoronaviruses. Based on sequence homology of the complete genome, we propose that these mustelid coronaviruses may be assigned to a new species. Comparison of FRCoV-NL-2010 with the partially sequenced ferret systemic coronavirus MSU-1 and ferret enteric coronavirus MSU-2 revealed that recombination in the spike, 3c and envelope genes occurred between different FRCoVs.

Coronaviruses in the Middle East Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness and kills about a third of people infected. The virus is common in dromedary camels, which can be a source of human infections. In a survey for MERSCoV in over 1300 Saudi Arabian camels, Sabir et al. found that dromedaries share three coronavirus species with humans. Diverse MERS lineages in camels have caused human infections, which suggests that transfer among host species occurs quite easily. Haagmans et al. made a MERS-CoV vaccine for use in camels, using poxvirus as a vehicle. The vaccine significantly reduced virus excretion, which should help reduce the potential for transmission to humans, and conferred cross-immunity to camelpox infections. Science , this issue p. 81 , p. 77

Background Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. Methods We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. Findings Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. Interpretation MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.

The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.

Highlights • A simple typing of MERS-CoV variants is crucial in monitoring the MERS-CoV outbreak. • The developed MERS-CoV typing assay provides an indication of the MERS-CoV variant. • It allows more informative initial screening in labs with basic sequencing capacity. • The data aid in informing effective international preparedness and response., Background Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human transmission occurs sporadically. The wide geographic distribution of MERS-CoV among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a MERS-CoV variant with efficient human-to-human transmission capabilities. Phylogenetic analysis of MERS-CoV has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples. Objective To develop a simple, reliable MERS-CoV variant typing assay to facilitate monitoring of MERS-CoV diversity in animals and humans. Study Design Phylogenetic analysis of presently known full-length MERS-CoV genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable MERS-CoV variant typing. RT-PCR assays targeting these regions were designed and optimized. Results A reverse-transcription PCR assay for MERS-CoV targeting a 615 bp spike fragment provides a phylogenetic clustering of MERS-CoV variants comparable to that of full-length genomes. The detection limit corresponds to a cycle treshold value of ∼35 with standard upE real time PCR assays on RNA isolated from MERS-CoV EMC. Nasal swabs from RT-PCR positive camels (Ct values 12.9–32.2) yielded reliable sequence information in 14 samples. Conclusions We developed a simple, reliable MERS-CoV variant typing assay which is crucial in monitoring MERS-CoV circulation in real time with relatively little investment on location.

To the Editor: Accumulating evidence indicates that dromedaries (Camelus dromedarius) are a reservoir for zoonotic transmission of Middle East respiratory syndrome coronavirus (MERS-CoV). Although numerous studies have looked at other livestock in the Middle East region, evidence for MERS-CoV infection has only been found in dromedaries (1). Extensive and continuous circulation of MERS-CoV occurs in the Al Shahaniya region of Qatar, most likely because of the presence of an international camel racing track and numerous barns holding camels (2,3). In April 2015, we investigated the MERS-CoV infection status of 15 healthy alpacas (Vicugna pacos) in a herd of 20 animals and 10 healthy dromedaries in a herd of 25 animals at a farm in this region (Technical Appendix). The herds were located at a distance of ≈200 m from each other within the barn complex and were cared for by the same animal workers, who lived in a common house between the herds at the complex. Both the alpacas and camels were kept as hobby animals. Serum samples were collected from all 25 animals. Nasal swabs were collected from all camels, whereas nasal, rectal, and oral swab specimens were collected only from a subset of the alpacas (Technical Appendix) because of logistical constraints. The serum samples were tested for IgG antibodies reactive with the S1 antigens of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV), and titers were calculated as described previously (4,5). MERS-CoV reactivity was confirmed by using a 90% plaque-reduction neutralization test (PRNT90) (3). Swab specimens were analyzed for MERS-CoV RNA by a screening PCR targeting the upE gene (6). MERS-CoV–specific antibodies were detected in all alpacas and all but 1 camel by protein microarray; reciprocal titers ranged from 49 to 773 for the alpacas and were >1,280 for the camels (Figure, panel A). PRNT90 testing confirmed the presence of MERS-CoV–specific antibodies; reciprocal neutralizing titers ranged from 80 to 320 for the alpacas and from 80 to >2,560 for 9 camels (Figure, panel B). All swab specimens were negative by PCR (Technical Appendix). None of the serum samples were reactive to SARS-CoV S1. The microarray was also conducted for bovine CoV and human CoV-229E antigens, which were used as a proxy for the serologically closely related dromedary betacoronavirus-1 HKU23 and 229E-related camelid alphacoronaviruses, respectively (7). Positive binding was detected for both antigens in alpaca and dromedary (data not shown). Figure Column scatterplots of MERS-CoV reactivity of serum samples from alpaca (n = 15) and dromedaries (n = 10) in the Al Shahaniya region of Qatar, April 2015. A) Plot of alpaca and dromedary serum titers of antibodies specific for S1 antigens of 2 coronaviruses ... Our observations prove the susceptibility of alpacas for natural MERS-CoV infection and lay the foundation for future studies to determine the potential of alpacas as another livestock reservoir for MERS-CoV. The alpacas in this study were the only alpacas in Qatar at the time and were located in a region where MERS-CoV is endemic. In a previous study, by using the same microarray technology, we found no evidence for MERS-CoV infection in alpacas from regions where MERS-CoV is not endemic (4). Although a study by Eckerle et al. demonstrated the potential of MERS-CoV to infect alpaca kidney cells in vitro (8) and alignment of mammalian DPP4 indicate that the 14 residues interacting with the MERS-CoV receptor binding domain of alpaca DPP4 are identical to that of dromedary DPP4 (Technical Appendix), the in vivo susceptibility of alpacas remained to be determined. The observed natural susceptibility of alpacas to MERS-CoV infection potentiates a broadening of the geographic range of MERS-CoV circulation to areas with large populations of alpacas. Alpacas are New World camelids, and the worldwide population of alpacas is estimated at 3 million animals, with ≈94% living in the high Andean regions of South America (Peru, Bolivia, Chile and Argentina), of which most are in Peru (constituting ≈88% of the world alpaca population) (http://lib.icimod.org/record/23682). Alpacas are increasingly being kept outside South America, mainly for their fleece, with estimated numbers in 2014 reaching 230,000 in the United States (http://lib.icimod.org/record/23682), 35,000 in the United Kingdom (http://www.bas-uk.com), and 150,000 in Australia (http://www.alpaca.asn.au). Although MERS-CoV has not been found in camelids other than dromedaries outside the Arabian Peninsula so far (9), our observations raise the question of whether other camelids could become infected if MERS-CoV were introduced to regions with large populations of alpacas and possibly other closely related camelids of the genera Lama, Vicugna, and Camelus. Because the date of infection of the alpacas and camels in this study is not known, we cannot speculate on the level of susceptibility of alpacas versus dromedaries based on the observed differences in antibody titers, which were lower in alpacas. It remains to be determined whether alpacas, in parallel with dromedaries, will actually shed MERS-CoV and are capable of independent maintenance of the virus in their population. Differences in susceptibility to viral pathogens between New and Old World camelids have been observed before (10). Therefore, understanding the risk requires further assessment of the reservoir competence of alpacas for MERS-CoV (e.g., through experimental infections) and an assessment of MERS-CoV–related viruses present in alpacas and other camelids in different parts of the world. Technical Appendix. Overview of background data and study results of alpaca and dromedary cohorts. Click here to view.(394K, pdf)

Graphical abstract MERS-CoV vaccine target groups and the desired vaccine induced immunological response for each group. I: camels; II: camel contacts; III-A: healthcare workers and patients; III-B: house-hold contacts and MERS cases., Highlights • MERS-CoV vaccines for dromedary camels and their human contacts can potentially stop virus transmission in the community. • Both neutralizing antibody and T-cell responses are required for protection • MERS-CoV vaccines should aim at inducing (long term) mucosal immunity in the different target populations. • The spike protein induces both neutralizing antibodies and T-cell responses and is the main target of the current vaccine candidates. • Epitope-based vaccines, focusing at (cross)-protective epitopes, could induce higher and/or broader responses., Middle East respiratory syndrome coronavirus (MERS-CoV) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. The re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. Ongoing efforts to develop MERS-CoV vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. Extending on our current knowledge of MERS, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish MERS-CoV transmission to humans. In addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next CoV outbreak.

We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels.

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.

Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3 , is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed “U-tube,” to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.

Aims: To identify the causative agent of the mortality in the fish, Mugil cephalus, in Muttukadu lagoon. Methods and Results: An enteric bacterium from the kidneys of moribund fish M. cephalus, was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis. Conclusions: The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus, found in Muttukadu lagoon. Significance and Impact of the Study: This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae, harbouring cationic factor, between human and fish may be of human health importance.

As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.

A new type of coronavirus has been identified as the causative agent underlying Middle East Respiratory Syndrome (MERS). The MERS coronavirus (MERS-CoV) has spread in the Middle East, but cases originating in the Middle East have also occurred in the European Union and the USA. Eight hundred and thirty-seven cases of MERS-CoV infection have been confirmed to date, including 291 deaths. MERS-CoV has infected dromedary camel populations in the Middle East at high rates, representing an immediate source of human infection. The MERS-CoV spike (S) protein, a characteristic structural component of the viral envelope, is considered as a key target of vaccines against coronavirus infection. In an initial attempt to develop a MERS-CoV vaccine to ultimately target dromedary camels, we constructed two recombinant adenoviral vectors encoding the full-length MERS-CoV S protein (Ad5.MERS-S) and the S1 extracellular domain of S protein (Ad5.MERS-S1). BALB/c mice were immunized with both candidate vaccines intramuscularly and boosted three weeks later intranasally. All the vaccinated animals had antibody responses against spike protein, which neutralized MERS-CoV in vitro. These results show that an adenoviral-based vaccine can induce MERS-CoV-specific immune responses in mice and hold promise for the development of a preventive vaccine that targets the animal reservoir, which might be an effective measure to eliminate transmission of MERS-CoV to humans. (C) 2014 Elsevier Ltd. All rights reserved.

Pathogen surveillance in animals does not provide a sufficient level of vigilance because it is generally confined to surveillance of pathogens with known economic impact in domestic animals and practically nonexistent in wildlife species. As most (re-) emerging viral infections originate from animal sources, it is important to obtain insight into viral pathogens present in the wildlife reservoir from a public health perspective. When monitoring living, free-ranging wildlife for viruses, sample collection can be challenging and availability of nucleic acids isolated from samples is often limited. The development of viral metagenomics platforms allows a more comprehensive inventory of viruses present in wildlife. We report a metagenomic viral survey of the Western Arctic herd of barren ground caribou (Rangifer tarandus granti) in Alaska, USA. The presence of mammalian viruses in eye and nose swabs of 39 free-ranging caribou was investigated by random amplification combined with a metagenomic analysis approach that applied exhaustive iterative assembly of sequencing results to define taxonomic units of each metagenome. Through homology search methods we identified the presence of several mammalian viruses, including different papillomaviruses, a novel parvovirus, polyomavirus, and a virus that potentially represents a member of a novel genus in the family Coronaviridae.

Summary Background Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe lower respiratory tract infection in people. Previous studies suggested dromedary camels were a reservoir for this virus. We tested for the presence of MERS-CoV in dromedary camels from a farm in Qatar linked to two human cases of the infection in October, 2013. Methods We took nose swabs, rectal swabs, and blood samples from all camels on the Qatari farm. We tested swabs with RT-PCR, with amplification targeting the E gene (upE), nucleocapsid ( N ) gene, and open reading frame (ORF) 1a. PCR positive samples were tested by different MERS-CoV specific PCRs and obtained sequences were used for phylogentic analysis together with sequences from the linked human cases and other human cases. We tested serum samples from the camels for IgG immunofluorescence assay, protein microarray, and virus neutralisation assay. Findings We obtained samples from 14 camels on Oct 17, 2013. We detected MERS-CoV in nose swabs from three camels by three independent RT-PCRs and sequencing. The nucleotide sequence of an ORF1a fragment (940 nucleotides) and a 4·2 kb concatenated fragment were very similar to the MERS-CoV from two human cases on the same farm and a MERS-CoV isolate from Hafr-Al-Batin. Eight additional camel nose swabs were positive on one or more RT-PCRs, but could not be confirmed by sequencing. All camels had MERS-CoV spike-binding antibodies that correlated well with the presence of neutralising antibodies to MERS-CoV. Interpretation Our study provides virological confirmation of MERS-CoV in camels and suggests a recent outbreak affecting both human beings and camels. We cannot conclude whether the people on the farm were infected by the camels or vice versa, or if a third source was responsible. Funding European Union projects EMPERIE (contract number 223498), ANTIGONE (contract number 278976), and the VIRGO consortium.

A novel coronavirus (CoV) that causes a severe lower respiratory tract infection in humans, emerged in the Middle East region in 2012. This virus, named Middle East respiratory syndrome (MERS)-CoV, is phylogenetically related to bat CoVs, but other animal species like dromedary camels may potentially act as intermediate hosts by spreading the virus to humans. Although human to human transmission has been demonstrated, analysis of human MERS clusters indicated that chains of transmission were not self-sustaining, especially when infection control was implemented. Thus, timely identification of new MERS cases followed by their quarantine, combined with measures to limit spread of the virus from the (intermediate) host to humans, may be crucial in controlling the outbreak of this emerging CoV.

[Background]: A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. [Methods]: We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. [Findings]: 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. [Interpretation]: MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection., RDS was funded by the European Public Health Training Program (Euphem), ECDC, Stockholm, Sweden. Contributions to the study were funded through the European Union FP7 projects EMPERIE (contract number 223498; to BLH, SLS, AO, CD) and ANTIGONE (contract number 278976; to CG, CD, MPGK, AO). Work in Bonn was also funded by Deutsche Forschungsgemeinschaft (DFG grant DR772/3-1 to CD).

A thorough understanding of virus diversity in wildlife provides epidemiological baseline information about pathogens. In this study, eye swab samples were obtained from semi-domesticated reindeer ( Rangifer tarandus tarandus) in Norway during an outbreak of infectious eye disease, possibly a very early stage of infectious keratoconjunctivitis (IKC). Large scale molecular virus screening, based on host nucleic acid depletion, sequence-independent amplification and next-generation sequencing of partially purified viral nucleic acid, revealed the presence of a new papillomavirus in 2 out of 8 eye swab samples and a new betaherpesvirus in 3 out of 8 eye swab samples collected from animals with clinical signs and not in similar samples in 9 animals without clinical signs. Whether either virus was responsible for causing the clinical signs or in any respect was associated to the disease condition remains to be determined.

RNA interference (RNAi) is a potential strategy to control shrimp viral diseases, including the white spot disease caused by White Spot Syndrome Virus (WSSV). Selection of genes for targeting is an important criterion. We have compared the efficacy of dsRNAs targeting structural (vp28 and vp281) and nonstructural genes (rr1 and dnapol) of WSSV in controlling viral multiplication in Penaeus monodon. Targeting the rr1 and vp28 genes provided better protection (93.3% and 90% survival respectively) compared to vp281 and dnapol in experimentally infected shrimp. Temporal transcriptional analysis of the corresponding genes and PCR-based diagnosis of WSSV in samples collected at different time points in the experiment supported this observation, thereby indicating that targeting a combination of rr1 and vp28 would be effective in limiting WSSV multiplication.

Koi herpesvirus, also known as cyprinid herpesvirus 3 (CyHV-3), is the aetiological agent of an emerging and mortal disease in common and koi carp. CyHV-3 virions present the characteristic morphology of other members of the order Herpesvirales, being composed of an envelope, a capsid containing the genome and a tegument. This study identified CyHV-3 structural proteins and the corresponding encoding genes using liquid chromatography tandem mass spectrometry-based proteomic approaches. In addition, exponentially modified protein abundance index analyses were used to estimate the relative abundance of the identified proteins in CyHV-3 virions. These analyses resulted in the identification of 40 structural proteins, which were classified based on bioinformatic analyses as capsid (three), envelope (13), tegument (two) and unclassified (22) structural proteins. Finally, a search for host proteins in purified CyHV-3 virions indicated the potential incorporation of up to 18 distinct cellular proteins. The identification of the proteins incorporated into CyHV-3 virions and determination of the viral genes encoding these proteins are key milestones for further fundamental and applied research on this virus.

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP -flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

To the Editor: Hepatitis E virus (HEV), a member of the family Hepeviridae and the genus Hepevirus, is transmitted by the fecal–oral route and causes liver inflammation, which leads to mortality rates of ≤20% in pregnant woman (1,2). Human hepatitis E is a major disease not only in developing countries but also in industrialized countries, and identification of animal strains of HEV in pigs and deer and its zoonotic potential has raised considerable public health concerns (1,3). Recent reports suggest that other animals such as rats, mongooses, chickens, rabbits, and trout also may harbor HEVs (1–5). The genomes of these viruses are ≈6.6 kb–7.2 kb and encode 3 open reading frames (ORFs) flanked by a capped 5′ end and a poly A tail at the 3′ end (1,3). We used random PCR amplification and high-throughput sequencing technology to investigate HEV sequences in ferrets (Mustela putorius) from the Netherlands. In 2010, fecal samples were collected from ferrets in the Netherlands and stored at −80°C. Samples that were negative for ferret coronavirus (6) were further characterized for other pathogens. Viral RNA was isolated and viral metagenomic libraries were constructed for 454 pyrosequencing as described (7,8), and 248,840 sequence reads were generated from 7 fecal samples. Using Blastn and Blastx (www.ncbi.nlm.nih.gov/BLAST), we identified 289 sequence reads in 1 sample that were related to rat HEV and that could be assembled into 6 contigs covering ≈50% of the ferret HEV (FRHEV) genome. We then developed a set of nested PCR primers on the basis of obtained sequences to detect viral RNA (Technical Appendix Table 1). Total RNA extracted from 43 ferret fecal samples collected from 19 locations in the Netherlands was used to perform reverse transcription PCR amplification. Using this PCR, we detected viral RNA in 4 (9.3%) fecal samples tested from 4 locations (distance between each sampling location ranged from 25 km to 127 km). All amplicons were confirmed by nucleotide sequencing. We have limited information regarding the clinical disease this virus may cause because these samples were obtained from household pet ferrets that did not show overt clinical signs. In addition, 4/16 animals from a single farm were IgG positive when tested for IgG against HEV by using recombinant human HEV protein (Wantai, Beijing, China). To characterize the complete genome of this virus, we selected 2 PCR-positive samples (FRHEV4 and FRHEV20), developed different sets of specific primers on the basis of sequence fragments obtained by 454 pyrosequencing, directly sequenced amplicons by Sanger sequencing, and used a rapid amplification of cDNA ends PCR to obtain 5′ and 3′ frame end sequences. Using overlapping fragments we assembled 2 complete FRHEV genome sequences that contained 6,854 nt, including a 13-nt 3′ poly A tail and a 12-nt 5′ end. FRHEV full-genome sequences FRHEV4 and FRHEV20 showed 98.6% sequence identity and were deposited into GenBank under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"JN998606","term_id":"397310723","term_text":"JN998606"}}JN998606 and {"type":"entrez-nucleotide","attrs":{"text":"JN998607","term_id":"397310728","term_text":"JN998607"}}JN998607, respectively. The FRHEV genome contains a complete ORF1 gene that encodes a nonstructural protein of 1,596 aa, an ORF2 gene that encodes a capsid protein of 654 aa, an ORF3 gene that encodes a phosphoprotein of 108 aa, and a 3′ noncoding region of 78 nt. Sequence analyses indicated that the FRHEV genome shared the highest identity (72.3%) with rat HEV. Sequence identity with HEV genotypes 1–4 and rabbit and avian HEVs ranged from 54.5% to 60.5% (Technical Appendix Table 2). The FRHEV genome organization was found to be slightly different from other HEVs and included a putative ORF (ORF4) of 552 nt that overlapped with ORF1 (Technical Appendix Figure). A similar pattern of genome organization was observed for both FRHEVs. Phylogenetic analysis of the complete genomes clearly showed that FRHEV was separated from genotype 1–4 HEVs and clustered with rat HEV (Figure). Similar phylogenetic clustering was observed when nucleotide and deduced amino acid sequences of ORF1, ORF2, and ORF3 were analyzed separately. The phylogenetic distance between rat HEV and FRHEV is larger than the distance between genotype 1 and genotype 2 HEV. Figure Phylogenetic tree based on the complete genomic sequences of ferret hepatitis E viruses (HEVs) and human, rabbit, swine, avian, and rat HEV strains. Names of HEV strains follow GenBank accession numbers. Sequence alignment was performed by using ClustalW ... In recent years, an increasing number of sporadic cases of hepatitis E have been reported (1,9). Several observations suggest that autochthonous cases are caused by zoonotic spread of infection from wild or domestic animals (1,3,9). In addition, IgG anti-HEV seropositivity in the United States has been associated with several factors, including having a pet at home (10). Further studies are needed to identify the zoonotic potential of FRHEV. Technical Appendix: Genome organization of hepatitis E viruses (HEVs) and initiation of translation of open reading frame 1 (ORF1), ORF2, and ORF3 of ferret HEV. Click here to view.(190K, pdf)

To the Editor: Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that can cause acute and chronic respiratory, enteric, and central nervous system disease in a variety of animal species (1). Recently, a novel ferret enteric CoV (FRECV) was indentified in domesticated ferrets (Mustela putorius) in which epizootic catarrhal enteritis had been diagnosed; the illness was characterized by foul-smelling green diarrhea with high mucus content, lethargy, anorexia, and vomiting (2). Another ferret CoV emerged in ferrets for which systemic pyogranulomatous inflammation, resembling the clinical and pathologic features of feline infectious peritonitis (FIP), was diagnosed (3–5). In 2010, we investigated the prevalence of CoV antibodies in 85 asymptomatic ferrets obtained from 1 ferret farm in the Netherlands. Previous studies have shown that antibodies against different members of the α-CoVs show broad cross-reactivity (6). We used FIP virus (FIPV)–infected cells to screen for CoV antibodies in an indirect immunoperoxidase assay. Because 32% of the ferret serum had a titer >20 in this assay, we concluded that these animals most likely had been exposed to a CoV. To test for a CoV in these animals, we analyzed RNA extracted from rectal swabs with a degenerate set of primers to amplify a conserved region within open reading frame (ORF) 1 of CoVs (7). Remarkably, 36 (42%) of samples tested were PCR positive, suggesting excretion of a CoV by a substantial proportion of ferrets tested. To corroborate that the CoV detected in the rectal swabs was a ferret CoV (FRCoV), we amplified and sequenced the nucleocapsid protein by using primer pair 5′-TCCCCGCGGGGCTGGCAACGGACAACGTGT-3′ and 5′-CCCAAGCTTTTAGTTTAGTTGACTAATAATTTCA-3′. Phylogenetic analyses of 2 of the sequences obtained indicated a variant nucleocapsid that was similar to other FRCoVs described previously but that did not group with 1 of these sequences directly (Figure). Amino acid alignment of 1 of these sequences (FRCoV-511c) with FRECV-MSU2 demonstrated 91.8% identity and 95.7% similarity, whereas this virus shows 89.3% identity and 95.2% similarity to ferret systemic CoV (FRSCV-MSU1). Figure Phylogenetic tree based on nucleotide sequences of the nucleocapsid (A) and spike gene (B) of ferret coronaviruses (FRCoVs) 4E98 (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"JF260916","term_id":"343482069","term_text":"JF260916"}} ... On the basis of obtained and published nucleocapsid sequences (2,4), we developed a TaqMan reverse transcription PCR to detect viral RNA using the following primers and degenerate probe: forward, 5′-TTGGAAAGAATGGTGCTAAAACTG-3′; reverse, 5′-CATTAGGCACGTTACCATCAAATT-3′; and probe, 5′-TAGGAACRCGTGGCACCAACCAA-3′. Using this more specific and sensitive assay, we detected viral RNA in 63% of the rectal swabs tested; other CoVs including FIPV, severe acute respiratory syndrome–CoV, and human CoV NL-63, were not amplified by this assay (data not shown). All samples that had tested positive in the ORF1-CoV PCR were confirmed positive with this TaqMan assay. To analyze FRCoV in ferrets from geographically distinct sites, we tested fecal samples from 90 animals without signs of disease (including epizootic catarrhal enteritis) from 39 different locations in the Netherlands. FRCoV nucleocapsid TaqMan and ORF1-CoV PCR demonstrated that 61% of the fecal samples and 72% of the locations were positive. Multiple testing of fecal swabs at different times and use of FRCoV-specific antibody assays would probably further increase the FRCoV prevalence rate. Further partial sequence analysis of the spike gene by using primers 5′-AARRTTAATGAGTGTGTGMGDTCA-3′ and 5′- CAACTCTYTTAAGCCARTCAAGG-3′ clearly showed that these viruses are more closely related to systemic FRCoVs than to FRECV (Figure). Amino acid alignment of 1 of these sequences (FRCoV-511c) with FRECV-MSU2 demonstrated 78% identity and 89% similarity, and FRCoV-511c shows 86% identity and 92% similarity to FRSCV-MSU1. After identification of severe acute respiratory syndrome CoV in humans in 2003 and related viruses in civet cats and bats, an increase in CoV surveillance in different animal species resulted in identification and characterization of a broad range of previously unrecognized CoVs (8). Here we report an enteric FRCoV circulating in the Netherlands in a high percentage of asymptomatic ferrets. The ferrets tested did not have a previous record of foul-smelling green diarrhea described previously to be associated with FRECV, a virus detected in the United States and further characterized in 2006 (2). On the basis of the phylogenetic analysis of the spike sequences, FRECV-MSU2 might have evolved through recombination with some other unknown CoV. Alternatively, the viruses isolated in the Netherlands grouped more closely with FRCoVs causing systemic disease (e.g., FRSCV-MSU1). Thus far, no evidence indicates that the animals testing positive for the enteric CoV showed clinical disease that pointed to pyogranulomatous inflammation, necrosis with or without perivasculitis, and vasculitis in abdominal and visceral organs associated with the systemic variant. Further genetic characterization of these enteric FRCoVs variants might show genetic differences that could explain the apparent pathotypes of these 2 FRCoVs. FRCoVs might evolve through mutation or deletion into viruses that cause systemic disease, or alternatively, different FRCoVs are circulating, analogous to the hypotheses put forward to explain the occurrence of FIP (9,10). Given the use of ferrets in testing efficacy of influenza virus vaccines and the propensity of CoVs to cross species barriers, further surveillance and investigation of the biology of these emerging FRCoVs is warranted.

Highlights • MERS-CoV genomes have been detected in nasal swabs of dromedaries. • Bat CoVs characterized thus far are relatively distantly related to MERS-CoV. • Routes of MERS-CoV zoonotic transmission are not well established. • Preventive measures should be taken to reduce zoonotic transmission of MERS-CoV., Coronaviruses (CoVs) able to infect humans emerge through cross-host transmission from animals. There is substantial evidence that the recent Middle East respiratory syndrome (MERS)-CoV outbreak is fueled by zoonotic transmission from dromedary camels. This is largely based on the fact that closely related viruses have been isolated from this but not any other animal species. Given the widespread geographical distribution of dromedaries found seropositive for MERS-CoV, continued transmission may likely occur in the future. Therefore, a further understanding of the cross host transmission of MERS-CoV is needed to limit the risks this virus poses to man.