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The rapid adaptation of SARS-CoV-2 within the host species and the increased viral transmission triggered the evolution of different SARS-CoV-2 variants. Though numerous monoclonal antibodies (mAbs) have been identified as prophylactic therapy for SARS-CoV-2, the ongoing surge in the number of SARS-CoV-2 infections shows the importance of understanding the mutations in the spike and developing novel vaccine strategies to target all variants. Here, we report the map of experimentally validated 74 SARS-CoV-2 neutralizing mAb binding epitopes of all variants. The majority (87.84%) of the potent neutralizing epitopes are localized to the receptor-binding domain (RBD) and overlap with each other, whereas limited (12.16%) epitopes are found in the N-terminal domain (NTD). Notably, 69 out of 74 mAb targets have at least one mutation at the epitope sites. The potent epitopes found in the RBD show higher mutations (4-10aa) compared to lower or modest neutralizing antibodies, suggesting that these epitopes might co-evolve with the immune pressure. The current study shows the importance of determining the critical mutations at the antibody recognition epitopes, leading to the development of broadly reactive immunogens targeting multiple SARS-CoV-2 variants. Further, vaccines inducing both humoral and cell-mediated immune responses might prevent the escape of SARS-CoV-2 variants from neutralizing antibodies.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein-based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43-positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.

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.

Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike (S) protein to bind to a host cell surface receptor. The rapid outbreak of emerging novel coronavirus, SARS-CoV 2 in China inculcates the need for the development of hasty and effective intervention strategies. Medicinal plants and natural compounds have been traditionally used to treat viral infections. Here, we generated VSV based pseudotyped viruses (pvs) of SARS-, MERS-, and SARS-2 CoVs to screen entry inhibitors from natural products. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind on their receptors and enter into the cells. SARS and MERS polyclonal antibodies neutralize SARSpv and SARS-2pv, and MERSpv respectively. Incubation of soluble ACE2 inhibited entry of SARS and SARS-2 pvs but not MERSpv. In addition, expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARSpv, SARS-2pv, and MERSpv respectively. Next, we showed the antiviral properties of known enveloped virus entry inhibitors, Spirulina and Green tea extracts against CoVpvs. SARSpv, MERSpv, and SARS-2pv entry were blocked with higher efficiency when preincubated with either green tea or spirulina extracts. Green tea provided a better inhibitory effect than the spirulina extracts by binding to the S1 domain of spike and blocking the interaction of spike with its receptor. Further studies are required to understand the exact mechanism of viral inhibition. In summary, we demonstrate that pseudotyped virus is an ideal tool for screening viral entry inhibitors. Moreover, spirulina and green tea could be promising antiviral agents against emerging viruses.

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.

Middle East respiratory syndrome coronavirus (MERS-CoV) still causes outbreaks despite public awareness and implementation of health care measures, such as rapid viral diagnosis and patient quarantine. Here we describe the current epidemiological picture of MERS-CoV, focusing on humans and animals affected by this virus and propose specific intervention strategies that would be appropriate to control MERS-CoV. One-third of MERS-CoV patients develop severe lower respiratory tract infection and succumb to a fatal outcome; these patients would require effective therapeutic antiviral therapy. Because of the lack of such intervention strategies, supportive care is the best that can be offered at the moment. Limiting viral spread from symptomatic human cases to health care workers and family members, on the other hand, could be achieved through prophylactic administration of MERS-CoV neutralizing antibodies and vaccines. To ultimately prevent spread of the virus into the human population, however, vaccination of dromedary camels – currently the only confirmed animal host for MERS-CoV – may be the best option to achieve a sustained drop in human MERS cases in time. In the end, a One Health approach combining all these different efforts is needed to tackle this zoonotic outbreak.

Hepatitis B virus (HBV) is genetically highly divergent and classified in ten genotypes and forty subgenotypes in distinct ethno-geographic populations worldwide. Ethiopia is a country with high HBV prevalence; however, little is known about the genetic variability of HBV strains that circulate. Here, we characterize the complete genome of 29 HBV strains originating from five Ethiopian regions, by 454 deep sequencing and Sanger sequencing. Phylogenetically, ten strains were classified as genotype A1 and nineteen as genotype D. Fifteen genotype D strains, provisionally named subgenotype D10, showed a novel distinct cluster supported by high bootstrap value and >4% nucleotide divergence from other known subgenotypes. In addition, the novel D10 strains harboured nine unique amino acid signatures in the surface, polymerase and X genes. Seventy-two per cent of the genotype D strains had the precore premature stop codon G1896A. In addition, 63% genotype A and 33% genotype D strains had the basal core promoter mutations, A1762T/G1764A. Furthermore, four pre-S deletion variants and two recombinants were identified in this study. In conclusion, we identified a novel HBV subgenotype D10 circulating in Ethiopia, underlining the high genetic variability of HBV strains in Africa.

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.

We characterized Middle East respiratory syndrome coronaviruses from a hospital outbreak in Jordan in 2015. The viruses from Jordan were highly similar to isolates from Riyadh, Saudi Arabia, except for deletions in open reading frames 4a and 3. Transmissibility and pathogenicity of this strain remains to be determined.

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.

Although hepatitis B virus (HBV) infection is hyperendemic in Ethiopia and constitutes a major public health problem, little is known about its genetic diversity, genotypes, and circulation. The aim of this study was to determine the molecular epidemiology and genetic diversity of HBV in Ethiopia, using 391 serum samples collected from HBsAg-positive blood donors living in five different geographic regions. The HBV S/pol gene was amplified, sequenced, and HBV genotypes, subgenotypes, serotypes, and major hydrophilic region (MHR) variants were determined. Phylogenetic analysis of 371 samples (95%) revealed the distribution of genotypes A (78%) and D (22%) in Ethiopia. Further phylogenetic analysis identified one subgenotype (A1) within genotype A, and 4 subgenotypes within genotype D (D1; 1.3%, D2; 55%, D4; 2.5%, and D6; 8.8%). Importantly, 24 isolates (30%) of genotype D formed a novel phylogenetic cluster, distinct from any known D subgenotypes, and two A/D recombinants. Analysis of predicted amino-acid sequences within the HBsAg revealed four serotypes: adw2 (79%), ayw1 (3.1%), ayw2 (7.8%), and ayw3 (11.6%). Subsequent examination of sequences showed that 51 HBV isolates (14%) had mutations in the MHR and 8 isolates (2.2%) in the reverse transcriptase known to confer antiviral resistance. This study provides the first description of HBV genetic diversity in Ethiopia with a predominance of subgenotypes A1 and D2, and also identified HBV isolates that could represent a novel subgenotype. Furthermore, a significant prevalence of HBsAg variants in Ethiopian population is revealed.

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.

A 31-year-old woman who was 10 weeks pregnant contracted Zika virus infection in Suriname; this led to fetal loss. ZIKV was detected in the woman's blood for at least 21 days.

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.

Current standard-of-care treatment of chronically infected hepatitis C virus (HCV) patients involves direct-acting antivirals (DAA). However, concerns exist regarding the emergence of drug -resistant variants and subsequent treatment failure. In this study, we investigate potential natural drug-resistance mutations in the NS5B gene of HCV genotype 1b from treatment-naïve patients. Population-based sequencing and 454 deep sequencing of NS5B gene were performed on plasma and liver samples obtained from 18 treatment- naïve patients. The quasispecies distribution in plasma and liver samples showed a remarkable overlap in each patient. Although unique sequences in plasma or liver were observed, in the majority of cases the most dominant sequences were shown to be identical in both compartments. Neither in plasma nor in the liver codon changes were detected at position 282 that cause resistance to nucleos(t)ide analogues. However, in 10 patients the V321I change conferring resistance to nucleos(t)ide NS5B polymerase inhibitors and in 16 patients the C316N/Y/H non-nucleoside inhibitors were found mainly in liver samples. In conclusion, 454-deep sequencing of liver and plasma compartments in treatment naïve patients provides insight into viral quasispecies and the pre-existence of some drug-resistant variants in the liver, which are not necessarily present in plasma.

The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells—which are important target cells in the development of congenital microcephaly—less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly., Recent Zika virus (ZIKV) infections have been associated with a range of neurological complications, in particular congenital microcephaly. Human neural progenitor cells (hNPCs) are thought to play an important role in the pathogenesis of microcephaly, and experimental ZIKV infection of hNPCs has been shown to induce cell death. However, the infection efficiency and rate of cell death have varied between studies, which might be related to intrinsic differences between African and Asian lineage ZIKV strains. Therefore, we determined the replication kinetics, including infection efficiency, burst size, and ability to induce cell death, of two Asian and two African ZIKV strains. African ZIKV strains replicated to higher titers in Vero cells, human glioblastoma (U87MG) cells, human neuroblastoma (SK-N-SH) cells, and hNPCs than Asian ZIKV strains. Furthermore, infection with Asian ZIKV strains did not result in significant cell death early after infection, whereas infection with African ZIKV strains resulted in high percentages of cell death in hNPCs. The differences between African and Asian lineage ZIKV strains highlight the importance of including relevant ZIKV strains to study the pathogenesis of congenital microcephaly and caution against extrapolation of experimental data obtained using historical African ZIKV strains to the current outbreak. Finally, the fact that Asian ZIKV strains infect only a minority of cells with a relatively low burst size together with the lack of early cell death induction might contribute to its ability to cause chronic infections within the central nervous system (CNS). IMPORTANCE The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells—which are important target cells in the development of congenital microcephaly—less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly.

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.

A longitudinal study of white spot syndrome virus (WSSV) infection and disease was conducted from January to August 2007 in a cluster of 12 small-holder shrimp ( Penaeus monodon ) ponds near Mallampudi in the Krishna District of Andhra Pradesh, India. Seven of the ponds had been sampled during the previous crop (Walker et al., 2011a); five adjacent ponds had not been sampled previously. Samples of mud were collected from the bottom of each pond at the commencement of the study. At intervals of approximately 10 days, farmed shrimp were sampled from the ponds and wild shrimp, crabs and plankton were sampled from inside ponds and from canals outside each pond. Of the 375 samples collected, 216 (57.6%) were WSSV-positive by Taqman PCR. The overall prevalence of WSSV infection varied significantly amongst sample types and was higher in farmed shrimp (76.0%) than for any other sample category (56% for crabs and plankton, 47% for wild shrimp, 25% for sediment). A wave of WSSV infection in plankton and wild crustaceans occurred across the study site, commencing at day 10, intensifying at day 20, subsiding at days 30 and 40, and passing by day 50. By day 60, only 2 ponds remained operational. WSSV-infection in plankton and wild crustacean samples collected from inside and outside these ponds again increased with a second peak at day 70. The pattern of WSSV infection in farmed shrimp appeared to follow the first wave of infection in plankton and wild crustaceans, with heavy viral genetic loads detected in most samples collected from days 30 to 80, including the final samples collected from 11 of the 12 ponds. Genotype analysis using the ORF94 variable number tandem repeat (VNTR) marker identified a very wide range of concurrently circulating WSSV genotypes (TRS1–TRS33) with multiple genotypes commonly detected in individual samples of all categories. Genotype TRS18, which had been associated with disease outbreaks in the previous crop, was not detected commonly in plankton or wild crustaceans. Although TRS18 was the most commonly detected genotype in farmed shrimp samples, it did not appear to be the cause of white spot disease outbreaks. There were several examples of simultaneous heavy infections with the same genotype in shrimp from several ponds but there was no clear pattern of association of a single WSSV genotype or a constellation of genotypes with disease outbreaks or pond abandonment.

A longitudinal study was conducted from January to August 2007 in a cluster of 61 small-holder shrimp (Penaeus monodon) ponds at Mallampudi (16°25′29″ N, 81°19′13 ″ E) in the Krishna District of Andhra Pradesh, India. Exhaustive PCR testing of postlarvae collected from ponds at the time of seeding detected no evidence of white spot syndrome virus (WSSV) infection. However, during grow-out, disease outbreaks occurred in 42 of the ponds (68.9%) in which the mean and median crop durations were 42.8 days and 40.5 days, respectively. Only three of the 61 ponds (4.9%) were harvested after more than 120 days of culture. Of 41 ponds sampled at harvest, 35 (85.4%) were WSSV-positive by PCR, including 27 of 28 (96.4%) disease outbreak ponds, of 17 which were graded as heavy or moderate infections. Eight of 13 (61.5%) normal harvest ponds sampled were WSSV-positive at the time of harvest, of which seven (53.8%) were graded as light or very light infections. WSSV genotype analysis was conducted on samples from 35 ponds using the ORF94 variable number tandem repeat (VNTR) marker. In total, 20 different genotypes from TRS1-TRS25 (1 to 25 repeats) were detected. Multiple TRS genotypes were detected in 27 of the 35 ponds sampled (77.1%) and 73 of the 146 individual shrimp sampled (50.0%). The most evident temporal and spatial associations of WSSV genotypes with disease outbreaks were the dominance of genotype TRS18 in seven ponds located on the eastern side and genotype TRS8 in eleven ponds in the central region of the study area. The study indicated a high risk of exposure to WSSV infection during grow-out and that multiple WSSV genotypes were circulating simultaneously in the farming area. The spatial and temporal pattern of WSSV genotype distribution suggested transmission of infection within two clusters of ponds.

A longitudinal study was conducted from January to August 2005 in small-holder black tiger shrimp (Penaeus monodon) ponds in the West Godavari District of Andhra Pradesh, India (16°25′ N, 81°19′ E). The study involved 457 ponds owned by low-income farmers participating in a better management practice (BMP) programme. Disease outbreaks occurred in 16.6% of ponds. There was significant spatial clustering of disease outbreaks with 31 (40.8%) of the 76 recorded disease outbreaks occurring in a single village block. Bivariate analysis indicated a 1.6-fold higher likelihood of disease outbreaks from nursery-stocked ponds but this was not significant in multivariate analysis due to the confounding effect of pond location. There was evidence of increasing prevalence of WSSV infection during grow-out. WSSV was detected in 5.9% of 119 batches of postlarvae tested at stocking, 38.2% of 34 juvenile batches collected at the time of transfer to grow-out ponds, and 47.0% of 336 pond stock tested at normal harvest or crop failure. WSSV was detected in 43 of 59 (72.9%) disease outbreak ponds tested and 115 of 277 (41.5%) non-outbreak ponds tested. Heavy WSSV infection was detected at harvest in 116 of the 336 (34.5%) of the ponds tested, including 78 ponds for which no outbreak was recorded. Duration of crop was recorded for 431 ponds with a mean of 117.0 days and a range of 20 to 176 days. Median duration was significantly shorter for disease outbreak ponds (68.5 days) compared to non-outbreak ponds (119.0 days). Duration of crop also varied according to WSSV detection levels at harvest, with median duration for ponds classified as heavy WSSV infection (108.5 days) significantly shorter than for ponds classified as either light WSSV infection (116.0 days) or WSSV-negative (116.5 days). The study indicated a high risk of WSSV infection during grow-out but a relatively low incidence of disease despite a high prevalence of heavy WSSV infection in non-outbreak ponds.

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.

Although hepatitis B virus (HBV) infection is hyperendemic in Ethiopia and constitutes a major public health problem, little is known about its genetic diversity, genotypes, and circulation. The aim of this study was to determine the molecular epidemiology and genetic diversity of HBV in Ethiopia, using 391 serum samples collected from HBsAg-positive blood donors living in five different geographic regions. The HBV S/pol gene was amplified, sequenced, and HBV genotypes, subgenotypes, serotypes, and major hydrophilic region (MHR) variants were determined. Phylogenetic analysis of 371 samples (95%) revealed the distribution of genotypes A (78%) and D (22%) in Ethiopia. Further phylogenetic analysis identified one subgenotype (A1) within genotype A, and 4 subgenotypes within genotype D (D1; 1.3%, D2; 55%, D4; 2.5%, and D6; 8.8%). Importantly, 24 isolates (30%) of genotype D formed a novel phylogenetic cluster, distinct from any known D subgenotypes, and two A/D recombinants. Analysis of predicted amino-acid sequences within the HBsAg revealed four serotypes: adw2 (79%), ayw1 (3.1%), ayw2 (7.8%), and ayw3 (11.6%). Subsequent examination of sequences showed that 51 HBV isolates (14%) had mutations in the MHR and 8 isolates (2.2%) in the reverse transcriptase known to confer antiviral resistance. This study provides the first description of HBV genetic diversity in Ethiopia with a predominance of subgenotypes A1 and D2, and also identified HBV isolates that could represent a novel subgenotype. Furthermore, a significant prevalence of HBsAg variants in Ethiopian population is revealed.

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.

In cidence of post-l arval mort alitie s of 30- 100% wa s reported from commercial freshwater prm'lln . I\llllero­ brachilfm rosellbergii (De Man) hatcheri es in Andhra Pradesh and Tamil Nadu (so uth-eastern states of In­ dia) sin ce 2001. Infec ted postlarvae (PL) ex h ibited clinical sy mptoms with lethargy, anorexia a nd whitening of abdominal muscles and thl! di sease was id entified as wh ite muscle di sease (WMD). The \vaterborn e infection of WMD was induced in the la­ boratory by exposing un infected and hea lthy M. ro­ senber{lii PL to th e IiItered muscle homogenates of the natuntlly infected PL, resultin g in mortality that reached 99% within 10 days post infec li on. Histo­ pathological examination of the infected a nimals revealed hi ghly necroti c Illll sculatu re. Degenerated muscl e areas showed aggregati ons of melanized nu­ clei. many of which looked like incl usion bodi es. Bac· te ri ological examination of affec ted PL showed t he presence of Staphylococcus sp p. as a predominant or­ gan i ~n1. whil e laboratory chall enge of hea lthy PL with this bacteri al isolate did not reproduce WlvlD.

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.

Hepatitis C virus (HCV) is genetically highly divergent and classified in seven major genotypes and approximately hundred subtypes. These genotypes/subtypes have different geographic distribution and response to antiviral therapy. In Ethiopia, however, little is known about their molecular epidemiology and genetic diversity. The aim of this study was to investigate the distribution and genetic diversity of HCV genotypes/subtypes in Ethiopia, using 49 HCV RNA positive samples. HCV genotypes and subtypes were determined based on the sequences of the core and the nonstructural protein 5B (NS5B) genomic regions. Phylogenetic analysis revealed that the predominant was genotype 4 (77.6%) followed by 2 (12.2%), 1 (8.2%), and 5 (2.0%). Seven subtypes were identified (1b, 1c, 2c, 4d, 4l, 4r and 4v), with 4d (34.7%), 4r (34.7%) and 2c (12.2%) as the most frequent subtypes. Consistent with the presence of these subtypes was the identification of a potential recombinant virus. One strain was typed as genotype 2c in the NS5B region sequence and genotype 4d in the core region. In conclusion, genotype 4 HCV viruses, subtypes 4d and 4r, are most prevalent in Ethiopia. This genotype is considered to be difficult to treat, thus, our finding has an important impact on the development of treatment strategies and patient management in Ethiopia.