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1.
Zhonghua Er Ke Za Zhi ; 57(8): 597-602, 2019 Aug 02.
Artigo em Chinês | MEDLINE | ID: mdl-31352744

RESUMO

Objective: To study the relationship between human cytomegalovirus (HCMV) envelope glycoprotein gene H and clinical features of children with congenital cytomegalovirus infection. Methods: A cohort study was conducted. Newborns diagnosed with congenital cytomegalovirus infection, hospitalized in the Department of Neonatology and Neonatal Intensive Care Unit (NICU) of the Children's Hospital, Zhejiang University School of Medicine, were included from July 2013 to December 2015.HCMV-DNA gH typing in urine, sputum or blood was conducted. Patients then were divided into gH1 group and gH2 group according to gH genotypes. Patients' data during hospitalization in newborn and 3-5 years of follow-up were collected.The relationships between gH genotype and clinical manifestations, laboratory examinations, hearing loss and neurological prognosis were analyzed by chi-square test, t test and non-parametric test. Results: A total of 21 cases were enrolled as congenital HCMV infection and followed-up for 3-5 years. Among them, 14 (67%) were gH1 type and 7 (33%) were gH2 type. No mixed infection was found. In the two groups, there were no significant differences in the ratio of males (9/14 vs. 3/7,P=0.397), or birth weight ((2 609±686) vs. (3 021±451) g, t=-1.436, P=0.167). Gestational age of gH1 group was younger than that of gH2 group (38 (29-40) vs. 39(38-40) weeks, Z=-2.18, P=0.029). Moderate to severe hearing loss detected by neonatal auditory brainstem response were found in 40 ears (20 cases). It was higher in gH1 group than that in gH2 group (4/22 vs.0/18, χ(2)=5.145, P=0.023). In the imaging examination of the nervous system, the Alarcon score of gH1 group was lower than that of gH2 group (0.4±0.3 vs. 1.3±1.1, t=-2.459,P=0.024).No significant statistical difference was found in the probability of motor or language development lag in gH2 group and gH1 group (4/7 vs.4/14, P=0.346). Conclusions: Compared with gH2 infection, gH1 infection in children has a younger gestational age. The major type of hearing loss in neonatal period is gH1 infection. Children with gH2 congenital infections are more likely to suffer from nervous systems damage.


Assuntos
Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/virologia , Citomegalovirus/classificação , Citomegalovirus/genética , Proteínas do Envelope Viral/genética , Criança , Estudos de Coortes , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/genética , Primers do DNA , Genótipo , Humanos , Recém-Nascido , Masculino
2.
Arch Virol ; 164(7): 1761-1770, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31065852

RESUMO

The distribution of hepatitis C virus (HCV) genotypes/subtypes varies among different populations. Here, we investigated HCV infection and its genotype distribution in injection drug users (IDUs) in Guangdong Province of China. A total of 318 IDUs from two prisons were recruited. The genotypes/subtypes of HCV in IDUs were determined by phylogenetic analysis using E1 and/or NS5B gene sequences. Our previous data on blood donors (BDs) with no history of drug use were used as control population data for comparison. Our results showed that the prevalence of HCV 3b (20.9% vs. 3.6%, P = 3.4E-9) and 6a (57.0% vs. 39.8%, P = 1.2E-5) was higher in IDUs than in BDs. In contrast, the prevalence of HCV 1b (43.4% vs. 5.6%, P = 9.8E-23) in BDs was higher than in IDUs. Phylogeographic analysis indicated that HCV 3b migrated from Yunnan to Guangdong Province and became endemic, with further transmission to other regions of China. The trend of HCV 3b dissemination in China in IDUs requires further attention, and a strategy for prevention and therapy is needed.


Assuntos
Usuários de Drogas/estatística & dados numéricos , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/epidemiologia , Adulto , Anticorpos Antivirais/sangue , China/epidemiologia , Feminino , Genótipo , Hepacivirus/isolamento & purificação , Hepatite C/transmissão , Humanos , Masculino , Filogeografia , Prisioneiros/estatística & dados numéricos , RNA Viral/genética , Abuso de Substâncias por Via Intravenosa , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética
3.
Mikrobiyol Bul ; 53(2): 144-155, 2019 Apr.
Artigo em Turco | MEDLINE | ID: mdl-31130119

RESUMO

Chronic hepatitis B (CHB) is an important public health problem affecting over 240 million people all around the world. The aim of the treatment in chronic hepatitis B is to prevent progression to cirrhosis and liver cancer. Interferons (standard and peginterferon) (Peg-IFN) and nucleoside/nucleotide analogues (NAs) are widely used in the treatment of CHB. The use of long-term therapy can however result in drug resistant mutations, which can lead to treatment failure. In patients with chronic hepatitis B, in addition to primary drug resistance mutations in the pol gene, compensatory mutations were reported. The genom of HBV polymerase (pol) gene overlaps with the envelope (S) gene. Nucleoside/nucleotide analogue (NA) resistance mutations in the pol gene of HBV, either from selection of primary or secondary resistance mutations, typically result in changes in HBsAg. Recent studies have conferred a new acronym for these HBV pol/S gene overlap mutants; ADAPVEMs, for antiviral drug-associated potential vaccine-escape mutants. The aim of this study was to investigate clinically and epidemiologically significant HBV pol/S gene mutations in NA treated CHB patients. In the study, a total of 100 patients who received nucleoside/nucleotide analogue therapy for one year or more were included. The levels of HBV DNA from serum samples were detected by the commercial real-time PCR assay and the mutations of pol/S genes by direct sequencing. Sixteen samples with low HBV DNA levels (> 200 IU/ml) could not be interpreted by sequencing due to insufficient amplification. Of the remaining 84 patients that could be sequenced HBV pol gene of HBV, 53 (63.09%) were males and 31 (36.91%) were women and the mean age was 47 ± 14.99 years (range: 20-67). Primary/secondary drug mutations (rtM204I/V, rtI169S, rtL180M, rtT184L, rtA194V, rtM204I/rtL91I, rtQ149K, rtQ215H/S, rtN238D) were detected in 38 (45.2%) of the patients. Because of the HBV pol/S gene overlapping, in 27 patients immun-selected amino acid substitutions (sI110L, sT127P, sS114A, sT123A), in nine patients HBIg selected escape mutants (sP120R, sT123N, sE164D, sY134F, sQ129H, sT118A, sP127K), in seven patients vaccine escape mutants (sT126I, sP120S, sG145A, s S193L) and in one patient misdiagnosis of HBsAg (sT131I) were detected. In addition, antiviral drug-associated potential vaccine-escape mutants were detected in 13 (15.4%) patients. In patients with chronic HBV, NAs including commonly used lamivudine were observed to have the potential for ADAPVEM to emerge during treatment. It was concluded that after determination of antiviral drug resistance and ADAPVEMs replanning of treatment should be done in the NA treatment of patients with CHB.


Assuntos
Antivirais , Farmacorresistência Viral , Produtos do Gene pol , Vírus da Hepatite B , Hepatite B Crônica , Mutação , Proteínas do Envelope Viral , Adulto , Idoso , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Feminino , Produtos do Gene pol/genética , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Humanos , Lamivudina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação/genética , Nucleotídeos/uso terapêutico , Proteínas do Envelope Viral/genética , Adulto Jovem
4.
Virol J ; 16(1): 59, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046787

RESUMO

BACKGROUND: Much evidence has demonstrated the influence of Hepatitis B virus (HBV) mutations on the clinical course of HBV infection. As large (L) protein plays a crucial role for viral entry, we hypothesized that mutations in the pre-S1 promoter region might affect the expression of L protein and subsequently change the biological characters of virus. METHODS: Patients infected with genotype C HBV were enrolled for analysis. HBV DNA sequences were inserted into a TA cloning vector and analyzed. To evaluate the effects of mutations in the pre-S1 promoter region, promoter activity and the expression of mRNA and L protein were analyzed using HepG2 cells. RESULTS: In total, 35 patients were enrolled and 13 patients (37.1%) had a single base substitution in the pre-S1 promoter region; the most frequent substitution was a G-to-A substitution at the 2765th base (G2765A) in the Sp1 region. The HBV viral load showed a negative correlation with the substitution ratio of the Sp1 region or G2765A (r = - 0.493 and - 0.473, respectively). Among those with a viral load ≤5.0 log IU/ml, patients with the G2765A substitution showed a significantly lower HBV viral load than those with the wild-type sequence. HepG2 cells transfected with the G2765A substitution vector showed reduced luciferase activity of the pre-S1 promoter, as well as reduced expression of pre-S1 mRNA and L protein. Furthermore, the G2765A substitution greatly reduced the L protein expression level of vector-produced virus particles. CONCLUSION: G2765A substitution in the pre-S1 promoter reduced the expression of L protein and resulted in a low viral load and less severe disease in chronic HBV infections.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas do Envelope Viral/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Feminino , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Carga Viral , Adulto Jovem
5.
Nat Commun ; 10(1): 2073, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061402

RESUMO

Isolation of broadly neutralizing human monoclonal antibodies (HmAbs) targeting the E2 glycoprotein of Hepatitis C virus (HCV) has sparked hope for effective vaccine development. Nonetheless, escape mutations have been reported. Ideally, a potent vaccine should elicit HmAbs that target regions of E2 that are most difficult to escape. Here, aimed at addressing this challenge, we develop a predictive in-silico evolutionary model for E2 that identifies one such region, a specific antigenic domain, making it an attractive target for a robust antibody response. Specific broadly neutralizing HmAbs that appear difficult to escape from are also identified. By providing a framework for identifying vulnerable regions of E2 and for assessing the potency of specific antibodies, our results can aid the rational design of an effective prophylactic HCV vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Proteínas do Envelope Viral/imunologia , Simulação por Computador , Desenho de Drogas , Mapeamento de Epitopos/métodos , Epitopos/genética , Epitopos/imunologia , Evolução Molecular , Hepacivirus/genética , Hepatite C/prevenção & controle , Hepatite C/virologia , Humanos , Modelos Biológicos , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/imunologia
6.
Virol J ; 16(1): 62, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068191

RESUMO

Dengue is hyperendemic in Colombia, where a cyclic behavior of serotype replacement leading to periodic epidemics has been observed for decades. This level of endemicity favors accumulation of dengue virus genetic diversity and could be linked to disease outcome. To assess the genetic diversity of dengue virus type 2 in Colombia, we sequenced the envelope gene of 24 virus isolates from acute cases of dengue or severe dengue fever during the period 2013-2016. The phylogenetic analysis revealed the circulation of the Asian-American genotype of dengue virus type 2 in Colombia during that period, the intra-genotype variability leading to divergence in two recently circulating lineages with differential geographic distribution, as well as the presence of nonsynonymous substitutions accompanying their emergence and diversification.


Assuntos
Vírus da Dengue/genética , Dengue/virologia , Variação Genética , Genótipo , RNA Viral/sangue , Adolescente , Adulto , Bancos de Espécimes Biológicos , Criança , Pré-Escolar , Colômbia/epidemiologia , Dengue/epidemiologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , RNA Viral/genética , Estudos Retrospectivos , Sorogrupo , Proteínas do Envelope Viral/genética , Adulto Jovem
7.
Ticks Tick Borne Dis ; 10(4): 935-941, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31072731

RESUMO

Tick-borne encephalitis virus (TBEV) is a member of the Flavivirus genus and is the main pathogenic arbovirus circulating in Europe, Russia and China. The envelope (E) protein is exposed on the viral surface and is the main antigen that is employed in diagnostic tests based on the detection of protein-specific antibodies from serum samples of infected individuals. The high degree of similarity among the E proteins of flaviviruses can, in some cases, lead to cross-reactivity and false-positive results in serological tests. Increased specificity in the detection of positive sera for different Flavivirus infections is often obtained by using a portion of the E protein, namely, the DIII domain. Different strategies and expression systems have been described for E and DIII protein production. Here, we present the optimization of an easy and fast method for TBEV E and DIII antigen production and partial purification from E. coli inclusion bodies. The antigenic properties of the produced antigens are retained, as validated by ELISAs with anti-TBEV murine sera as well as sera from infected human patients. The potential applications of both proteins as diagnostic reagents were confirmed.


Assuntos
Antígenos Virais/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/biossíntese , Antígenos Virais/genética , Clonagem Molecular , Encefalite Transmitida por Carrapatos/diagnóstico , Encefalite Transmitida por Carrapatos/imunologia , Ensaio de Imunoadsorção Enzimática , Infecções por Flavivirus/diagnóstico , Infecções por Flavivirus/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética
8.
Virol J ; 16(1): 69, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133031

RESUMO

BACKGROUND: Coronaviruses (CoVs) primarily cause enzootic infections in birds and mammals but, in the last few decades, have shown to be capable of infecting humans as well. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and, more recently, Middle-East respiratory syndrome (MERS) has demonstrated the lethality of CoVs when they cross the species barrier and infect humans. A renewed interest in coronaviral research has led to the discovery of several novel human CoVs and since then much progress has been made in understanding the CoV life cycle. The CoV envelope (E) protein is a small, integral membrane protein involved in several aspects of the virus' life cycle, such as assembly, budding, envelope formation, and pathogenesis. Recent studies have expanded on its structural motifs and topology, its functions as an ion-channelling viroporin, and its interactions with both other CoV proteins and host cell proteins. MAIN BODY: This review aims to establish the current knowledge on CoV E by highlighting the recent progress that has been made and comparing it to previous knowledge. It also compares E to other viral proteins of a similar nature to speculate the relevance of these new findings. Good progress has been made but much still remains unknown and this review has identified some gaps in the current knowledge and made suggestions for consideration in future research. CONCLUSIONS: The most progress has been made on SARS-CoV E, highlighting specific structural requirements for its functions in the CoV life cycle as well as mechanisms behind its pathogenesis. Data shows that E is involved in critical aspects of the viral life cycle and that CoVs lacking E make promising vaccine candidates. The high mortality rate of certain CoVs, along with their ease of transmission, underpins the need for more research into CoV molecular biology which can aid in the production of effective anti-coronaviral agents for both human CoVs and enzootic CoVs.


Assuntos
Coronavirus/química , Proteínas do Envelope Viral/química , Animais , Coronavirus/genética , Coronavirus/patogenicidade , Infecções por Coronavirus/virologia , Humanos , Vírus da SARS/química , Vírus da SARS/genética , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/genética , Zoonoses/transmissão , Zoonoses/virologia
9.
Soft Matter ; 15(22): 4525-4540, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31099376

RESUMO

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.


Assuntos
HIV-1/química , Manose/química , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/química , Proteínas do Envelope Viral/química , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Manose/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Nanopartículas Metálicas/química , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
10.
Microb Cell Fact ; 18(1): 70, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971255

RESUMO

BACKGROUND: Bacterial surface display systems were developed to surface expose heterologous proteins or peptides for different applications, such as peptide libraries screening and live bacterial vaccine design. Various outer membrane proteins, such as outer membrane protein A (OmpA), OmpC and outer membrane pore protein E precursor (PhoE), have been used as carriers for surface display, fused to the proteins or peptides of interest in Gram-negative bacteria. Here, we investigated the utility of constitutively expressed OmpF for the display of foreign immune epitopes on the Escherichia coli cell surface and then compared it with plasmid-induced expression of OmpF and OmpC. RESULTS: Enhanced expression of OmpF was linked to a mutation in the OmpF promoter sequence. This mutation rendered OmpF an ideal carrier protein for the enriched display of a target of interest on the bacterial surface. To this end, we grafted two peptides, harboring important epitopes of the hepatitis B virus (HBV) S antigen and human papilloma virus (HPV) L2 protein, onto OmpF of E. coli by genome editing. The resultant fused OmpF proteins were constitutively expressed in the edited E. coli and purified by membrane component extraction. The epitope that displayed on the bacterial surface was verified by SDS-PAGE, western blotting, flow cytometry, and immunoelectron microscopy of the intact bacteria. We further compared this constitutive expression with plasmid-induced expression of OmpF and OmpC in bacterial cells using the same methods for verification. We found that plasmid-induced expression is much less efficient than constitutive expression of OmpF from the bacterial genome. CONCLUSIONS: Enhanced expression of OmpF in a plasmid-independent manner provides an amenable way to display epitopes on the bacterial surface and sheds light on ways to engineer bacteria for biotechnological applications.


Assuntos
Técnicas de Visualização da Superfície Celular , Epitopos/genética , Porinas/genética , Anticorpos Antibacterianos , Proteínas do Capsídeo/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Edição de Genes , Proteínas Oncogênicas Virais/genética , Plasmídeos/genética , Mutação Puntual , Proteínas do Envelope Viral/genética
11.
Retrovirology ; 16(1): 9, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940160

RESUMO

BACKGROUND: We previously showed that the gM of HSV-1 could restrict the release of infectious HIV-1 from cells. In this study, we analyzed if the four HSV-1 glycoproteins (gD, gB, and gH/gL), which are the minimum glycoproteins required for HSV-1 entry, restricted the release of infectious HIV-1. RESULTS: Of these four glycoproteins, gD and gH/gL restricted the production of infectious HIV-1 from cells transfected with an infectious molecular clone of HIV-1 (strain NL4-3) while gB had no significant effect. Pulse-chase analyses indicated that gD did not affect the biosynthesis and processing of gp160 into gp120/gp41, the transport of the gp120/gp41 to the cell surface, or the release of HIV-1 particles from the cell surface. Our analyses revealed that gD was incorporated into HIV-1 virus particles while gp120/gp41 was excluded from released virus particles. Truncated mutants of gD revealed that the cytoplasmic domain was dispensable but that a membrane bound gD was required for the restriction of release of infectious HIV-1. Finally, cell lines expressing gD also potently restricted the release of infectious virus. CONCLUSIONS: Due to its ability to exclude HIV-1 gp120/gp41 from maturing virus, gD may provide a useful tool in deciphering mechanisms of Env incorporation into maturing virus particles.


Assuntos
HIV-1/fisiologia , Herpesvirus Humano 1/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Linhagem Celular , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/metabolismo , Herpesvirus Humano 1/genética , Humanos , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética
12.
Arch Virol ; 164(7): 1753-1760, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31025116

RESUMO

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.


Assuntos
Baculoviridae/genética , Proteínas do Capsídeo/biossíntese , Tymovirus/crescimento & desenvolvimento , Tymovirus/genética , Proteínas do Envelope Viral/biossíntese , Montagem de Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Vírus Chikungunya/genética , Expressão Gênica/genética , Lycopersicon esculentum/virologia , Mariposas/citologia , Proteínas do Envelope Viral/genética
13.
Vet Microbiol ; 232: 30-41, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030842

RESUMO

The lineage 3 of porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) was first reported in mainland China in 2010 and it has spread rapidly in recent years. Here, two novel lineage 3 strains of PRRSV-2 were isolated from diseased pigs in Southwestern China during 2017-2018, and were designated as GZgy17 and SCya18. The complete genomes of the two isolates were then determined, and sequence alignment revealed that GZgy17 had the same discontinuous 30-amino acid (aa) deletion in NSP2 as JXA1, while SCya18 contained the discontinuous 131-aa deletion in NSP2 identical to that of NADC30, when compared to the strain VR-2332. Notably, GZgy17 contained an additional 19-aa deletion in NSP2, and SCya18 had a unique 3-nt deletion in its 3'UTR. Homology and phylogenetic analysis showed that GZgy17 and SCya18 shared low nucleotide homology (91.2-92.0%) with QYYZ and were classified into a new cluster of lineage 3 strains based on ORF5 genotyping. Recombination analyses revealed that GZgy17 and SCya18 both originated from a SH/CH/2016-like (lineage 3) strain and had recombined with a JXA1-like (lineage 8) and a NADC30-like (lineage 1) strain, respectively. Furthermore, we compared the virulence of the two strains in 4-week-old piglets. The results showed that GZgy17 caused mortality rates of 20% and exhibited higher pathogenicity in piglets compared to SCya18. Our findings suggest that recombination might be responsible for the variations in pathogenicity of lineage 3 strains of PRRSV-2 and highlight the importance of surveillance of this lineage in China.


Assuntos
Genoma Viral , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus Reordenados/isolamento & purificação , Recombinação Genética , Animais , China , Evolução Molecular , Variação Genética , Filogenia , Síndrome Respiratória e Reprodutiva Suína/mortalidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Suínos , Proteínas do Envelope Viral/genética , Virulência
14.
Vet Microbiol ; 232: 79-83, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030849

RESUMO

Classical swine fever virus (CSFV) envelope glycoprotein Erns has been shown to bind to cell surface sulphated-heparin-like glycosaminoglycans (GAGs), which participate in cell attachment of the virus. In this study, the CSFV Erns gene was codon optimized for expression in the yeast Pichia pastoris. A C-terminally truncated Erns recombinant protein lacking the previously identified heparin-binding domain (HBD) bound to heparin column, suggesting the presence of another HBD in CSFV Erns. Sequence analyses of the CSFV Erns coding region revealed a common potential N-terminal HBD at residues 301-311. Site-directed mutagenesis of the basic amino acids at K303 and K306 significantly reduced the heparin-binding affinity of the protein. Further mutations of both T310 and H311 had little effect. Thus, a novel potential heparin-binding site near the N-terminus of CSFV strain TD96 Erns has been detected, and the two basic amino acids K303 and K306 are crucial for binding activity to heparin matrix and cell-surface GAGs.


Assuntos
Vírus da Febre Suína Clássica/química , Glicoproteínas/química , Heparina/metabolismo , Proteínas do Envelope Viral/química , Aminoácidos/metabolismo , Sítios de Ligação , Vírus da Febre Suína Clássica/genética , Glicoproteínas/genética , Mutagênese Sítio-Dirigida , Mutação , Fases de Leitura Aberta , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas do Envelope Viral/genética
15.
Arch Virol ; 164(6): 1697-1703, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30968212

RESUMO

Disease caused by bovine leukemia virus (BLV) results in significant economic losses to the livestock industry. To date, there is only one report describing the strains found in Italy. BLV strains (n = 24), collected between 2012 and 2016 from four different Italian regions, were genetically analyzed by direct sequencing of a portion of the BLV env gene, and the sequences were compared with those in the GenBank database. The Italian BLVs clustered into genotypes G2, G4, G6, G7, and G8, revealing a high level of BLV genetic heterogeneity in Italy. This study provides a basis for further investigations into the evolutionary relationship between BLV strains.


Assuntos
Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/genética , Análise de Sequência de RNA/métodos , Proteínas do Envelope Viral/genética , Animais , Bovinos , Evolução Molecular , Variação Genética , Técnicas de Genotipagem , Itália , Vírus da Leucemia Bovina/isolamento & purificação , Filogenia
16.
Arch Virol ; 164(5): 1323-1334, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877450

RESUMO

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.


Assuntos
Circovirus/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Macrófagos Alveolares/patologia , Resposta a Proteínas não Dobradas/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Infecções por Circoviridae/patologia , Infecções por Circoviridae/veterinária , Retículo Endoplasmático/virologia , Endorribonucleases/metabolismo , Macrófagos Alveolares/virologia , Microscopia Eletrônica de Transmissão , Suínos , Doenças dos Suínos , Proteínas do Envelope Viral/metabolismo , eIF-2 Quinase/metabolismo
17.
Arch Virol ; 164(5): 1371-1382, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30888564

RESUMO

Equine herpesvirus 1 (EHV-1) induces serious respiratory infections, viral abortion, neurological signs, and neonatal mortality in horses. Despite the use of vaccines, EHV-1 infection also causes a high annual economic burden to the equine industry. The poor immunogenicity of and protection conferred by EHV-1 vaccines are the major factors responsible for the spread of EHV-1 infection. The present study examined the immunogenicity of a novel DNA vaccine co-expressing FliC, a flagellin protein, in Salmonella abortus equi and the gD protein of EHV-1. Mice and horses were immunized intramuscularly with the vaccine, and mice were challenged with EHV-1. Immunofluorescence and western blotting revealed that FliC and gD can be efficiently expressed in cells. This novel vaccine significantly increased gD-specific antibody and interferon gamma (IFN-γ) levels in immunized mice and horses. Compared with controls, the viral load and morbidity were markedly reduced in FliC-gD-immunized mice after they were challenged with EHV-1. Furthermore, the immunogenicity of FliC-gD in a natural host was tested. Our results indicate that vaccinated mice and horses exhibit increased humoral and improved cellular immune responses.


Assuntos
Anticorpos Antivirais/sangue , Flagelina/imunologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/imunologia , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Linhagem Celular , Feminino , Flagelina/genética , Células HEK293 , Infecções por Herpesviridae/imunologia , Cavalos , Humanos , Imunoglobulina G/sangue , Interferon gama/sangue , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Salmonella/imunologia , Receptor 5 Toll-Like/metabolismo , Proteínas do Envelope Viral/genética , Carga Viral
18.
J Biotechnol ; 295: 41-48, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30826446

RESUMO

The recent Ebola virus disease (EVD) outbreaks make the development of efficacious and low cost vaccines against Ebola virus (EBOV) an urgent goal. Multiepitopic vaccines allow a rational design rendering vaccines able to induce proper immune responses in terms of polarization and potency. In addition, the pathogen variants can be easily covered by including epitopes conserved among relevant isolates. Other important aspects to consider in vaccination are the costs associated to production, distribution, and administration of the vaccine. Plants provide an advantageous platform for this purpose, since they yield biomass at very low costs and some species can be used to formulate purification-free oral vaccines. In the present study, a multiepitopic protein called Zerola, which carries epitopes from the EBOV glycoprotein (GP), was designed based on immunoinformatic approaches and current experimental evidence on B cell protective GP epitopes. Moreover, expression studies performed in nuclear-transformed tobacco lines confirmed the capacity of the plant cell to synthetize the Zerola antigenic protein. The generation of this plant-based candidate vaccine is a step forward in the development of highly efficient and low cost EBOV vaccines.


Assuntos
Vacinas contra Ebola , Ebolavirus/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes , Proteínas do Envelope Viral , Células Cultivadas , Vacinas contra Ebola/química , Vacinas contra Ebola/genética , Vacinas contra Ebola/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Células Vegetais , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
19.
Int J Med Sci ; 16(3): 355-365, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30911269

RESUMO

Dengue virus belongs to the Flaviviridae family which also includes viruses such as the Zika, West Nile and yellow fever virus. Dengue virus generally causes mild disease, however, more severe forms of the dengue virus infection, dengue haemorrhagic fever (DHF) and dengue haemorrhagic fever with shock syndrome (DSS) can also occur, resulting in multiple organ failure and even death, especially in children. The only dengue vaccine available in the market, CYD-TDV offers limited coverage for vaccinees from 9-45 years of age and is only recommended for individuals with prior dengue exposure. A number of mutations that were shown to attenuate virulence of dengue virus in vitro and/or in vivo have been identified in the literature. The mutations which fall within the conserved regions of all four dengue serotypes are discussed. This review hopes to provide information leading to the construction of a live attenuated dengue vaccine that is suitable for all ages, irrespective of the infecting dengue serotype and prior dengue exposure.


Assuntos
Vacinas contra Dengue/farmacologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Capsídeo/química , Ensaios Clínicos Fase III como Assunto , Dengue/prevenção & controle , Vacinas contra Dengue/imunologia , Genoma Viral , Humanos , Mutação , Vacinas Atenuadas/farmacologia , Vacinas Sintéticas/farmacologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Virulência , Fatores de Virulência/química , Fatores de Virulência/genética
20.
PLoS Negl Trop Dis ; 13(3): e0007212, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30845254

RESUMO

Zika virus (ZIKV) is a mosquito-borne positive sense RNA virus. Recently, ZIKV emerged into the Western hemisphere as a human health threat, with severe disease associated with developmental and neurological complications. The structural envelope protein of ZIKV and other neurotropic flaviviruses contains an extended CD-loop relative to non-neurotropic flaviviruses, and has been shown to augment ZIKV stability and pathogenesis. Here we show that shortening the CD-loop in ZIKV attenuates the virus in mice, by reducing the ability to invade and replicate in the central nervous system. The CD-loop mutation was genetically stable following infection in mice, though secondary site mutations arise adjacent to the CD-loop. Importantly, while shortening of the CD-loop attenuates the virus, the CD-loop mutant maintains antigenicity in immunocompetent mice, eliciting an antibody response that similarly neutralizes both the mutant and wildtype ZIKV. These findings suggest that the extended CD-loop in ZIKV is a determinant of neurotropism and may be a target in live-attenuated vaccine design, for not only ZIKV, but for other neurotropic flaviviruses.


Assuntos
Sistema Nervoso Central/virologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Zika virus/patogenicidade , Animais , Anticorpos Antivirais/imunologia , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Virulência , Replicação Viral/genética , Zika virus/genética
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