Erratum for Sun et al., “Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis”

ABSTRACT Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV. IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.

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A Single Mutation at Position 156 in the Envelope Protein of Tembusu Virus Is Responsible for Virus Tissue Tropism and Transmissibility in Ducks

Journal of Virology ◽

10.1128/jvi.00427-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 12

Author(s):

Dawei Yan ◽

Ying Shi ◽

Haiwang Wang ◽

Guoxin Li ◽

Xuesong Li ◽

...

Keyword(s):

Amino Acid ◽

Virus Replication ◽

Envelope Protein ◽

Tissue Tropism ◽

Single Mutation ◽

Mosquito Vectors ◽

E Protein ◽

Virus West Nile ◽

Tembusu Virus ◽

Domain I

ABSTRACT Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses, such as Japanese encephalitis virus, West Nile virus, and Bagaza virus, is able to transmit vector-independently. To date, why these flaviviruses can be transmitted without mosquito vectors remains poorly understood. To explore the key molecular basis of flavivirus transmissibility, we compared virus replication and transmissibility of an early and a recent TMUV in ducks. The recent TMUV strain FX2010 replicated systemically and transmitted efficiently in ducks, while the replication of early strain MM1775 was limited and did not transmit among ducks. The TMUV envelope protein and its domain I were responsible for tissue tropism and transmissibility. The mutation S156P in the domain I resulted in disruption of N-linked glycosylation at amino acid 154 of the E protein and changed the conformation of “150 loop” of the E protein, which reduced virus replication in lungs and abrogated transmission in ducks. These data indicate that the 156S in the envelope protein is critical for TMUV tissue tropism and transmissibility in ducks in the absence of mosquitos. Our findings provide novel insights on understanding TMUV transmission among ducks. IMPORTANCE Tembusu virus, similar to other mosquito-borne flaviviruses such as WNV, JEV, and BAGV, can be transmitted without the presence of mosquito vectors. We demonstrate that the envelope protein of TMUV and its amino acid (S) at position 156 is responsible for tissue tropism and transmission in ducks. The mutation S156P results in disruption of N-linked glycosylation at amino acid 154 of the E protein and changes the conformation of “150 loop” of the E protein, which induces limited virus replication in lungs and abrogates transmission between ducks. Our findings provide new knowledge about TMUV transmission among ducks.

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A critical amino acid residue, Asp446, in UDP-glucuronosyltransferase

Biochemical Journal ◽

10.1042/bj3250587 ◽

1997 ◽

Vol 325 (3) ◽

pp. 587-591 ◽

Cited By ~ 13

Author(s):

Hidetomo IWANO ◽

Hiroshi YOKOTA ◽

Satoru OHGIYA ◽

Naomi YOTUMOTO ◽

Akira YUASA

Keyword(s):

Amino Acid ◽

Amino Acid Residue ◽

Amino Acid Substitution ◽

Critical Role ◽

Enzymic Activity ◽

Male Rat ◽

Transferase Activity ◽

Rt Pcr ◽

Taq Polymerase ◽

Udp Glucuronosyltransferase

An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from Ysh A1337GG1384A had no transferase activity. Two other mutant cDNAs with Ysh A1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and Ysh G1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from Ysh G1384A (named Ysh V462M) exhibited enzymic activity, but the one from Ysh A1337G (named Ysh D446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme.

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Amino Acid Substitution(s) in the Stem-Anchor Region of Langat Virus Envelope Protein Attenuates Mouse Neurovirulence

Virology ◽

10.1006/viro.2001.0959 ◽

2001 ◽

Vol 286 (1) ◽

pp. 54-61 ◽

Cited By ~ 12

Author(s):

Michael R. Holbrook ◽

Haolin Ni ◽

Robert E. Shope ◽

Alan D.T. Barrett

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Envelope Protein ◽

Virus Envelope ◽

Langat Virus ◽

Virus Envelope Protein

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Molecular, Antigenic, and Functional Characteristics of Ferric Enterobactin Receptor CfrA in Campylobacter jejuni

Infection and Immunity ◽

10.1128/iai.00666-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5437-5448 ◽

Cited By ~ 31

Author(s):

Ximin Zeng ◽

Fuzhou Xu ◽

Jun Lin

Keyword(s):

Amino Acid ◽

Campylobacter Jejuni ◽

Growth Promotion ◽

Stress Hormones ◽

Critical Role ◽

Basic Amino Acid ◽

Inhibitory Effect ◽

Amino Acid Identity ◽

Pcr Analysis ◽

Immunoblotting Analysis

ABSTRACTThe ferric enterobactin receptor CfrA not only is responsible for high-affinity iron acquisition inCampylobacter jejunibut also is essential forC. jejunicolonization in animal intestines. In this study, we determined the feasibility of targeting the iron-regulated outer membrane protein CfrA for immune protection againstCampylobactercolonization. Alignment of complete CfrA sequences from 15Campylobacterisolates showed that the levels of amino acid identity for CfrA range from 89% to 98%. Immunoblotting analysis using CfrA-specific antibodies demonstrated that CfrA was dramatically induced under iron-restricted conditions and was widespread and produced in 32Campylobacterprimary strains from various sources and from geographically diverse areas. The immunoblotting survey results were highly correlated with the results of an enterobactin growth promotion assay and a PCR analysis usingcfrA-specific primers. Inactivation of thecfrAgene also impaired norepinephrine-mediated growth promotion, suggesting that CfrA is required forC. jejunito sense intestinal stress hormones during colonization. Complementation of thecfrAmutant with a wild-typecfrAallele intransfully restored the production and function of CfrA. A growth assay using purified anti-CfrA immunoglobulin G demonstrated that specific CfrA antibodies could block the function of CfrA, which diminished ferric enterobactin-mediated growth promotion under iron-restricted conditions. The inhibitory effect of CfrA antibodies was dose dependent. Immunoblotting analysis also indicated that CfrA was expressed and immunogenic in chickens experimentally infected withC. jejuni. Amino acid substitution mutagenesis demonstrated that R327, a basic amino acid that is highly conserved in CfrA, plays a critical role in ferric enterobactin acquisition inC. jejuni.Together, these findings strongly suggest that CfrA is a promising vaccine candidate for preventing and controllingCampylobacterinfection in humans and animal reservoirs.

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