Warm winters are associated to more intense West Nile virus circulation in southern Spain.

West Nile virus (WNV) is the most widely distributed mosquito-borne flavivirus in the world. This flavivirus can infect humans causing in some cases a fatal neurological disease and birds are the main reservoir hosts. WNV is endemic in Spain, and human cases have been reported since 2004. Although different studies analyse how climatic conditions can affect the dynamics of WNV infection, very few use long-term datasets. Between 2003 and 2020 a total of 2,724 serum samples from 1,707 common coots (Fulica atra) were analysed for the presence of WNV-specific antibodies. Mean (SD) annual seroprevalence was 24.67% (0.28) but showed high year-to-year variations ranging from 5.06% (0.17) to 68.89% (0.29). Significant positive correlations (p < 0.01) were observed between seroprevalence and maximum winter temperature and mean spring temperature. The unprecedented WNV outbreak in humans in the south of Spain in 2020 was preceded by a prolonged period of escalating WNV local circulation. Given current global and local climatic trends, WNV circulation is expected to increase in the next decades. This underscores the necessity of implementing One Health approaches to reduce the risk of future WNV outbreaks in humans. Our results suggest that higher winter and spring temperatures may be used as an early warning signal of more intense WNV circulation among wildlife in Spain, and consequently highlight the need of more intense vector control and surveillance in human inhabited areas.

Rhizobia induce SYMRK endocytosis in Phaseolus vulgaris root hair cells.

MAIN CONCLUSION: PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor. Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

SARS-CoV-2-Mediated Lung Edema and Replication Are Diminished by Cystic Fibrosis Transmembrane Conductance Regulator Modulators.

Coronaviruses (CoVs) of genera α, ß, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

Wide-field ESD for Barrett's adenocarcinoma at the gastroesophageal junction: technical approaches to facilitate en bloc R0 resection.

Video 1Wide-field ESD for Barrett's adenocarcinoma.

A One Health view of the West Nile virus outbreak in Andalusia (Spain) in 2020.

Reports of West Nile virus (WNV) associated disease in humans were scarce in Spain until summer 2020, when 77 cases were reported, eight fatal. Most cases occurred next to the Guadalquivir River in the Sevillian villages of Puebla del Río and Coria del Río. Detection of WNV disease in humans was preceded by a large increase in the abundance of Culex perexiguus in the neighbourhood of the villages where most human cases occurred. The first WNV infected mosquitoes were captured approximately one month before the detection of the first human cases. Overall, 33 positive pools of Cx. perexiguus and one pool of Culex pipiens were found. Serology of wild birds confirmed WNV circulation inside the affected villages, that transmission to humans also occurred in urban settings and suggests that virus circulation was geographically more widespread than disease cases in humans or horses may indicate. A high prevalence of antibodies was detected in blackbirds (Turdus merula) suggesting that this species played an important role in the amplification of WNV in urban areas. Culex perexiguus was the main vector of WNV among birds in natural and agricultural areas, while its role in urban areas needs to be investigated in more detail. Culex pipiens may have played some role as bridge vector of WNV between birds and humans once the enzootic transmission cycle driven by Cx. perexiguus occurred inside the villages. Surveillance of virus in mosquitoes has the potential to detect WNV well in advance of the first human cases.

MERS-CoV ORF4b is a virulence factor involved in the inflammatory pathology induced in the lungs of mice.

No vaccines or specific antiviral drugs are authorized against Middle East respiratory syndrome coronavirus (MERS-CoV) despite its high mortality rate and prevalence in dromedary camels. Since 2012, MERS-CoV has been causing sporadic zoonotic infections in humans, which poses a risk of genetic evolution to become a pandemic virus. MERS-CoV genome encodes five accessory proteins, 3, 4a, 4b, 5 and 8b for which limited information is available in the context of infection. This work describes 4b as a virulence factor in vivo, since the deletion mutant of a mouse-adapted MERS-CoV-Δ4b (MERS-CoV-MA-Δ4b) was completely attenuated in a humanized DPP4 knock-in mouse model, resulting in no mortality. Attenuation in the absence of 4b was associated with a significant reduction in lung pathology and chemokine expression levels at 4 and 6 days post-infection, suggesting that 4b contributed to the induction of lung inflammatory pathology. The accumulation of 4b in the nucleus in vivo was not relevant to virulence, since deletion of its nuclear localization signal led to 100% mortality. Interestingly, the presence of 4b protein was found to regulate autophagy in the lungs of mice, leading to upregulation of BECN1, ATG3 and LC3A mRNA. Further analysis in MRC-5 cell line showed that, in the context of infection, MERS-CoV-MA 4b inhibited autophagy, as confirmed by the increase of p62 and the decrease of ULK1 protein levels, either by direct or indirect mechanisms. Together, these results correlated autophagy activation in the absence of 4b with downregulation of a pathogenic inflammatory response, thus contributing to attenuation of MERS-CoV-MA-Δ4b.

Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells.

Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

Visualization of the Crossroads between a Nascent Infection Thread and the First Cell Division Event in Phaseolus vulgaris Nodulation.

The development of a symbiotic nitrogen-fixing nodule in legumes involves infection and organogenesis. Infection begins when rhizobia enter a root hair through an inward structure, the infection thread (IT), which guides the bacteria towards the cortical tissue. Concurrently, organogenesis takes place by inducing cortical cell division (CCD) at the infection site. Genetic analysis showed that both events are well-coordinated; however, the dynamics connecting them remain to be elucidated. To visualize the crossroads between IT and CCD, we benefited from the fact that, in Phaseolus vulgaris nodulation, where the first division occurs in subepidermal cortical cells located underneath the infection site, we traced a Rhizobium etli strain expressing DsRed, the plant cytokinesis marker YFP-PvKNOLLE, a nuclear stain and cell wall auto-fluorescence. We found that the IT exits the root hair to penetrate an underlying subepidermal cortical (S-E) cell when it is concluding cytokinesis.

A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies.

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.

Genetically Engineered Live-Attenuated Middle East Respiratory Syndrome Coronavirus Viruses Confer Full Protection against Lethal Infection.

There are no approved vaccines against the life-threatening Middle East respiratory syndrome coronavirus (MERS-CoV). Attenuated vaccines have proven their potential to induce strong and long-lasting immune responses. We have previously described that severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a virulence factor. Based on this knowledge, a collection of mutants carrying partial deletions spanning the C-terminal domain of the E protein (rMERS-CoV-E*) has been generated using a reverse genetics system. One of these mutants, MERS-CoV-E*Δ2in, was attenuated and provided full protection in a challenge with virulent MERS-CoV after a single immunization dose. The MERS-CoV-E*Δ2in mutant was stable as it maintained its attenuation after 16 passages in cell cultures and has been selected as a promising vaccine candidate.IMPORTANCE The emergence of the new highly pathogenic human coronavirus SARS-CoV-2 that has already infected more than 80 million persons, killing nearly two million of them, clearly indicates the need to design efficient and safe vaccines protecting from these coronaviruses. Modern vaccines can be derived from virus-host interaction research directed to the identification of signaling pathways essential for virus replication and for virus-induced pathogenesis, in order to learn how to attenuate these viruses and design vaccines. Using a reverse genetics system developed in our laboratory, an infectious cDNA clone of MERS-CoV was engineered. Using this cDNA, we sequentially deleted several predicted and conserved motifs within the envelope (E) protein of MERS-CoV, previously associated with the presence of virulence factors. The in vitro and in vivo evaluation of these deletion mutants highlighted the relevance of predicted linear motifs in viral pathogenesis. Two of them, an Atg8 protein binding motif (Atg8-BM), and a forkhead-associated binding motif (FHA-BM), when deleted, rendered an attenuated virus that was evaluated as a vaccine candidate, leading to full protection against challenge with a lethal dose of MERS-CoV. This approach can be extended to the engineering of vaccines protecting against the new pandemic SARS-CoV-2.

Middle East Respiratory Syndrome Coronavirus Gene 5 Modulates Pathogenesis in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pneumonia that emerged in 2012. There is limited information on MERS-CoV pathogenesis, as data from patients are scarce and the generation of animal models reproducing MERS clinical manifestations has been challenging. Human dipeptidyl peptidase 4 knock-in (hDPP4-KI) mice and a mouse-adapted MERS-CoV strain (MERSMA-6-1-2) were recently described. hDPP4-KI mice infected with MERSMA-6-1-2 show pathological signs of respiratory disease, high viral titers in the lung, and death. In this work, a mouse-adapted MERS-CoV infectious cDNA was engineered by introducing nonsynonymous mutations contained in the MERSMA-6-1-2 genome into a MERS-CoV infectious cDNA, leading to a recombinant mouse-adapted virus (rMERS-MA) that was virulent in hDDP4-KI mice. MERS-CoV adaptation to cell culture or mouse lungs led to mutations and deletions in genus-specific gene 5 that prevented full-length protein expression. In contrast, analysis of 476 MERS-CoV field isolates showed that gene 5 is highly stable in vivo in both humans and camels. To study the role of protein 5, two additional viruses were engineered expressing a full-length gene 5 (rMERS-MA-5FL) or containing a complete gene 5 deletion (rMERS-MA-Δ5). rMERS-MA-5FL virus was unstable, as deletions appeared during passage in different tissue culture cells, highlighting MERS-CoV instability. The virulence of rMERS-MA-Δ5 was analyzed in a sublethal hDPP4-KI mouse model. Unexpectedly, all mice died after infection with rMERS-MA-Δ5, in contrast to those infected with the parental virus, which contains a 17-nucleotide (nt) deletion and a stop codon in protein 5 at position 108. Expression of interferon and proinflammatory cytokines was delayed and dysregulated in the lungs of rMERS-MA-Δ5-infected mice. Overall, these data indicated that the rMERS-MA-Δ5 virus was more virulent than the parental one and suggest that the residual gene 5 sequence present in the mouse-adapted parental virus had a function in ameliorating severe MERS-CoV pathogenesis.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus causing human infections with high mortality rate (∼35%). Animal models together with reverse-genetics systems are essential to understand MERS-CoV pathogenesis. We developed a reverse-genetics system for a mouse-adapted MERS-CoV that reproduces the virus behavior observed in humans. This system is highly useful to investigate the role of specific viral genes in pathogenesis. In addition, we described a virus lacking gene 5 expression that is more virulent than the parental one. The data provide novel functions in IFN modulation for gene 5 in the context of viral infection and will help to develop novel antiviral strategies.

Rhizobium etli CE3-DsRed pMP604: a useful biological tool to study initial infection steps in Phaseolus vulgaris nodulation.

MAIN CONCLUSION: Rhizobium etli CE3-DsRed pMP604 drives infection 12-24 h earlier than R. etli CE3-DsRed and it is an excellent tool in live-cell imaging studies of IT developement in P. vulgaris roots. The study of the cellular dynamics of nodulation has frequently been limited by the difficulty of performing live-cell imaging in nodule primordia and legume roots, which are constituted by multiple cell layers, such is the case of Phaseolus vulgaris. Seeking conditions to reduce the time it takes for rhizobia to infect P. vulgaris root, we decided to explore the nodulation properties of Rhizobium etli CE3 pMP604, a strain that constitutively produces Nod factors through a flavonoids-independent transcriptional activation which is often used to purify Nod factors. Even though the strain infects 12-24 h earlier than the parental R. etli CE3 strain, infection thread (IT) formation, nodule organogenesis processes and N2-fixation activity are similar for both strains. Additionally, we have confirmed that R. etli CE3-DsRed pMP604 is an excellent tool to trace IT development in P. vulgaris roots.

Mycobacterium avium complex infection in a patient with systemic sclerosis- associated interstitial lung disease: A case report.

We describe a case of a 65-year-old male with recently diagnosed diffuse cutaneous systemic sclerosis associated with usual interstitial pneumonia and pulmonary hypertension. Patient presented to the emergency department complaining of low-grade fever, increased sputum production, progressive dyspnea and weight loss. High-resolution computed tomography scan showed multifocal bronchiectasis with multiple small nodules. Bronchoalveolar lavage culture was positive for Mycobacterium intracellulare. Antimicrobial treatment was started which improved respiratory symptoms. One month after the initiation of antibiotics, cyclophosphamide therapy was started with adequate tolerance.

Adsorción de Cd(II) y Pb(II) presentes en solución acuosa con hueso de nanche (Byrsonima crassifolia) / Adsorption of Cd(ll) and Pb(ll) present in aqueous solution with nanche stone (Byrsonima crassifolia) / Adsorção de Cd(ll) e Pb(ll) presente em solução aquosa com osso nanche (Byrsonima crassifolia)

Resumen El hueso de nanche es un residuo agrícola con gran potencial de uso como adsorbente. Al respecto, se evaluó la capacidad de adsorción de iones Cd(II) y Pb(II), presentes en solución acuosa, utilizando este residuo. Las pruebas realizadas en este estudio fueron: la determinación del punto de carga cero (pHPZC), determinación de sitios activos (método de Boehm y espectroscopía FTIR) y el desarrollo de los modelos matemáticos de Langmuir, Freundlich y Prausnitz-Radke, a través de isotermas de adsorción. Los resultados obtenidos del pHPZC del hueso de nanche estuvieron en un rango ácido (6,0), mientras que la concentración de sitios ácidos y básicos fue de 0,1037 y 0,046 mol/g, respectivamente. Los espectros infrarrojos (FTIR) detectaron sitios funcionales ácidos asociados al grupo fenol, ácidos carboxílicos y lactonas. Finalmente, la capacidad de adsorción del hueso de nanche para Cd(II) y Pb(II) se incrementó con el pH y alcanzó porcentajes de remoción hasta de 84 % para Cd(II) a pH 8, y de 82% para Pb(II) a pH 5. En conclusión, el pH y la presencia de sitios funcionales ácidos fueron determinantes en la eliminación de los iones Pb(II) y Cd(II).

Abstract The nanche stone is an agricultural residue that has great potential as adsorbent. Then, the adsorption capacity of Cd(II) and Pb(II) ions present in aqueous solution was evaluated, using the stone as adsorbent. The tests performed for this purpose were the determination of point of zero charge (pHPZC), determination of active sites (Boehm method and FTIR spectroscopy), and the development of the mathematical models of Langmuir, Freundlich, and Prausnitz-Radke through adsorption isotherms. The results obtained from the pHPZC of the nanche stone were in an acidic range (6,0), while the concentration of acid and basic sites were 0.1037 and 0.046 mol/g respectively. The infrared spectra (FTIR) detected acid functional sites associated with the phenol group, carboxylic acids, and lactones. Finally, the adsorption capacity of nanche stone to Cd(II) and Pb(II), increased with the pH, achieving percentages of removal up to 84% for Cd (II) to pH 8, and of 82% for Pb(II) to pH 5. In conclusion, the pH and the presence of acidic functional sites were decisive in the elimination of the Pb(II) and Cd(II) ions.

Resumo O osso Nanche é um resíduo agrícola que possui grande potencial como adsorvente, neste respeito, a capacidade de adsorção de íons Cd(II) e Pb(II) presentes em solução aquosa foi avaliada, utilizando osso como adsorvente. Os testes realizados para este fim foram a determinação do ponto zero de carga (pHPZC), a determinação de sites ativos (método Boehm e espectroscopia FTIR) e o desenvolvimento dos modelos matemáticos de Langmuir, Freundlich e Prausnitz-Radke através de isotermas de adsorção. Os resultados obtidos do pHPZC do osso nanche foram em uma faixa ácida (6,0), enquanto a concentração de ácidos e sites básicos foram 0,1037 e 0,046 mol / g, respectivamente. Os espectros de infravermelho (FTIR) detectaram sites funcionais ácidos associados ao grupo fenol, ácidos carboxílicos e lactonas. Finalmente, a capacidade de adsorção do osso nanche para Cd(II) e Pb(II), aumentou com o pH, atingindo porcentagens de remoção de até 84% para o Cd(II) em pH 8, e 82% para Pb(II) a pH 5. Em conclusão, o pH e a presença de sites funcionais ácidos foram decisivos na eliminação dos íons Pb (II) e Cd (II).

Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein.

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.

Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries-apart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV-infected dromedaries, we identified several MERS-CoV-specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.

Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice.IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.