Bicluster pattern of codon context usages between flavivirus and vector mosquito Aedes aegypti: relevance to infection and transcriptional response of mosquito genes

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias in usages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis-driven tests to examine the role of codon context bias in evolution of vector–virus interactions at the molecular level.     

Susceptibility of Florida Aedes aegypti and Aedes albopictus to dengue viruses from Puerto Rico

Locally acquired dengue cases in the continental U.S. are rare. However, outbreaks of dengue‐1 during 2009, 2010, and 2013 in Florida and dengue‐1 and −2 in Texas suggest vulnerability to transmission. Travel and commerce between Puerto Rico and the U.S. mainland is common, which may pose a risk for traveler‐imported dengue cases. Mosquitoes were collected in Florida and used to evaluate their susceptibility to dengue viruses (DENV) from Puerto Rico. Aedes aegypti and Ae. albopictus were susceptible to virus infection with DENV‐1 and −2. No significant differences were observed in rates of midgut infection or dissemination between Ae. aegypti or Ae. albopictus for DENV‐1 (6–14%). Aedes aegypti was significantly more susceptible to midgut infection with DENV‐2 than Ae. albopictus (Ae. aegypti, ∼28%; Ae. albopictus, ∼9%). The dissemination rate with dengue‐2 virus for Ae. aegypti (23%) was greater than Ae. albopictus (0%), suggesting that Ae. albopictus is not likely to be an important transmitter of the DENV‐2 isolate from Puerto Rico. These results are discussed in light of Florida's vulnerability to DENV transmission.     

Cell membrane enolase of Aedes albopictus C6/36 cells is involved in the entrance mechanism of dengue virus (DENV)

Currently, there are no antiviral drugs that effectively reduce the risks and treat the symptoms associated with dengue virus (DENV). Consequently, efforts remain primarily focused on transmission reduction. One such effort concerns DENV receptors in mosquito vectors Aedes aegypti and Aedes albopictus. Despite a lack of direct evidence demonstrating the binding of DENV to cells in mosquito vectors, one putative DENV binding protein has been α-enolase. To develop a deeper understanding, this study tested whether DENV proteins bind to enolase localized in the cytoplasmic membrane of C6/36 cells using both anti-enolase-specific antibodies, and by colocalization analysis, using confocal microscopy.Additionally, to probe the interaction of enolase with the DENV E protein, we performed a docking analysis using PatchDock and FireDock software packages. Study results demonstrate that the DENV E protein interacts with enolase in the plasma membrane of C6/36 cells of Ae. albopictus. Specific anti-enolase antibodies were found to inhibit DENV infection of these cells. Moreover, enolase was found to be localized to the cytoplasmic membrane, cytoplasm, and nucleus. These combined findings suggest that enolase participates in the entrance mechanism of DENV into vector cells.     

The Effect of Multiple Evolutionary Selections on Synonymous Codon Usage of Genes in the Mycoplasma bovis Genome

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.     

The Native Wolbachia Symbionts Limit Transmission of Dengue Virus in Aedes albopictus

Background

The chikungunya (CHIK) outbreak that struck La Reunion Island in 2005 was preceded by few human cases of Dengue (DEN), but which surprisingly did not lead to an epidemic as might have been expected in a non-immune population. Both arboviral diseases are transmitted to humans by two main mosquito species, Aedes aegypti and Aedes albopictus. In the absence of the former, Ae. albopictus was the only species responsible for viral transmission on La Reunion Island. This mosquito is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB. While Wolbachia does not affect replication of CHIK virus (CHIKV) in Ae. albopictus, a similar effect was not observed with DEN virus (DENV).

Methods/Principal Findings

To understand the weak vectorial status of Ae. albopictus towards DENV, we used experimental oral infections of mosquitoes from La Reunion Island to characterize the impact of Wolbachia on DENV infection. Viral loads and Wolbachia densities were measured by quantitative PCR in different organs of Ae. albopictus where DENV replication takes place after ingestion. We found that: (i) Wolbachia does not affect viral replication, (ii) Wolbachia restricts viral density in salivary glands, and (iii) Wolbachia limits transmission of DENV, as infectious viral particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after the infectious blood-meal.

Conclusions

We show that Wolbachia does not affect the replication of DENV in Ae. albopictus. However, Wolbachia is able to reduce viral infection of salivary glands and limit transmission, suggesting a role of Wolbachia in naturally restricting the transmission of DENV in Ae. albopictus from La Reunion Island. The extension of this conclusion to other Ae. albopictus populations should be investigated.     

Evolution of endemic and sylvatic lineages of dengue virus

Recent disease outbreaks have raised awareness of tropical pathogens, especially mosquito-borne viruses. Dengue virus (DENV) is a widely studied mammalian pathogen transmitted by various species of mosquito in the genus Aedes, especially Aedes aegypti and Aedes albopictus. The prevailing view of the research community is that endemic viral lineages that cause epidemics of DENV in humans have emerged over time from sylvatic viral lineages, which persist in wild, non-human primates. These notions have been examined by researchers through phylogenetic analyses of the envelope gene (E) from the four serotypes of DENV (serotypes DENV-1 to DENV-4). In these previous reports, researchers used visual inspection of a phylogeny in order to assert that sylvatic lineages lead to endemic clades. In making this assertion, these researchers also reasserted the model of periodic sylvatic to endemic disease outbreaks. Since that study, there has been a significant increase in data both in terms of metadata (e.g., place and host of isolation) and genetic sequences of DENV. Here, we re-examine the model of sylvatic to endemic shifts in viral lineages through a phylogenetic tree search and character evolution study of metadata on the tree. We built a dataset of nucleotide sequences for 188 isolates of DENV that have metadata on sylvatic or endemic sampling along with three orthologous sequences from West Nile virus as the outgroup for the phylogenetic analysis. In contrast to previous research, we find that there are several shifts from endemic to sylvatic lineages as well as sylvatic to endemic lineages, indicating a much more dynamic model of evolution. We propose that a model that allows oscillation between sylvatic and endemic hosts better captures the dynamics of DENV transmission.     

Zika Virus,a New Threat for Europe?

BackgroundSince its emergence in 2007 in Micronesia and Polynesia, the arthropod-borne flavivirus Zika virus (ZIKV) has spread in the Americas and the Caribbean, following first detection in Brazil in May 2015. The risk of ZIKV emergence in Europe increases as imported cases are repeatedly reported. Together with chikungunya virus (CHIKV) and dengue virus (DENV), ZIKV is transmitted by Aedes mosquitoes. Any countries where these mosquitoes are present could be potential sites for future ZIKV outbreak. We assessed the vector competence of European Aedes mosquitoes (Aedes aegypti and Aedes albopictus) for the currently circulating Asian genotype of ZIKV.Conclusions/SignificanceIn combination with the restricted distribution of European Ae. albopictus, our results on vector competence corroborate the low risk for ZIKV to expand into most parts of Europe with the possible exception of the warmest regions bordering the Mediterranean coastline.     

Epidemiological evidence of mosquito-borne viruses among persons and vectors in Iran: A study from North to South

Arthropod-borne viruses are a group of the most important emerging pathogens. They cause a range of diseases in vertebrate hosts and threaten human health (Gan and Leo, 2014). The global distribution of arboviruses is associated with the vector which is strongly affected by changes in environmental conditions. Dengue virus (DENV) and Chikungunya virus (CHIKV), which cause high annual infected cases and have an increasing geographic distribution, are transmitted by Aedes spp. mosquitoes, in particular Ae. albopictus and Ae. Aegypti (Presti et al., 2014; Higuera and Ramírez, 2018). Although, the main vector of dengue virus, Ae. aegypti, was not detected in Iran, other possible important vectors such as Ae. Albopictus and Ae. unilineatus were recorded (Doosti et al., 2016; Yaghoobi-Ershadi et al., 2017). West Nile virus (WNV), a member of the genus Flaviviruses, is one of the most widespread arboviruses (Chancey et al., 2015). The epidemiological evidence of WNV in different hosts in Iran was found (Bagheri et al., 2015), and the circulation of WNV in the main vector, Culex pipiens s.l. and Cx. pipiens, has been proved (Shahhosseini et al., 2017). Due to limited information on the situation of CHIKV, DENV and WNV in Iran, we performed a wide geographical investigation to determine the prevalence of IgG specific antibodies in human samples as well as the genome of WNV, CHIKV and DENV in mosquitoes.     

Comparative genome sequence analysis of Sulfolobus acidocaldarius and 9 other isolates of its genus for factors influencing codon and amino acid usage

In the present study, major constraints for codon and amino acid usage of Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodali, Sulfolobus islandis and 6 other isolates from islandicus species of genus Sulfolobus were investigated. Correspondence analysis revealed high significant correlation between the major trend of synonymous codon usage and gene expression level, as assessed by the “Codon Adaptation Index” (CAI). There is a significant negative correlation between Nc (Effective number of codons) and CAI demonstrating role of codon bias as an important determinant of codon usage. The significant correlation between major trend of synonymous codon usage and GC3s (G + C at third synonymous position) indicated dominant role of mutational bias in codon usage pattern. The result was further supported from SCUO (synonymous codon usage order) analysis. The amino acid usage was found to be significantly influenced by aromaticity and hydrophobicity of proteins. However, translational selection which causes a preference for codons that are most rapidly translated by current tRNA with multiple copy numbers was not found to be highly dominating for all studied isolates. Notably, 26 codons that were found to be optimally used by genes of S. acidocaldarius at higher expression level and its comparative analysis with 9 other isolates may provide some useful clues for further in vivo genetic studies on this genus.     

Codon usage bias and evolutionary analyses of Zika virus genomes

Zika virus (ZIKV) is a member of the family Flaviviridae and contains a single-stranded RNA genome with positive-polarity. Like Dengue, Zika virus uses Aedes aegypti mosquito as a vector to infect human with a wide range of clinical signs, from asymptomatic to influenza-like syndrome. Despite significant progress in genomic analyses, how a viral relationship with two different hosts affect the overall fitness, constancy, and dodging of hosts immune system are elusive. Here we analyzed Zika virus codon-based evolution using eleven strains from different geographical locations. The overall codon usage was similar and slightly bias among all strains. An occurrence of A-ending in highly-preferred codons and analysis by various approaches strongly suggests that mutational bias is the main force shaping codon usage in this virus. However, natural selection and geographical realities cannot be ignored in marginal influence on codon usage. The viral genomes naturally favor Aedes aegypti over human host for tRNA pool in translation. Such findings will assist researchers in understanding elements contribute to viral adaptation and evolutionary setup with hosts.     

Genome-wide analysis of codon usage bias patterns in an enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 strain

Enterogenic Escherichia coli (ETEC) F18 strains are the main pathogenic bacteria causing severe diarrhea in humans and domestic animals. However, the information about synonymous codon usage pattern of ETEC F18 genome remains unclear. We conducted a genome-wide analysis of synonymous codon usage patterns in the ETEC F18 strain SRA: SAMN02471895. After filtering of the complete genome sequence, 4327 coding sequences were analyzed using multivariate statistical methods to calculate synonymous codon usage patterns and to evaluate the influence of various factors in shaping the codon usage. The mean GC content was 51.38%, with a slight preference for G/C-ending codons. Twenty-two codons were determined as ‘‘optimal codons”. ENC plots showed some of the genes were on or close to the expected curve, while only points with low-ENC values were below the curve. PR2 analysis showed that GC and AT were not used proportionally, suggesting major roles for mutational pressure and natural selection in shaping usage. Neutrality plots showed a significant correlation between GC12 and GC3, suggesting that mutational pressure is responsible for nucleotide composition in shaping the strength of codon usage. Translational selection was the main factor shaping the codon usage pattern of ETEC F18 genome, while other factors such as protein length, GRAVY and ARO values also influenced codon usage to some extent. We analyzed the codon usage pattern systematically and identified the factors shaping codon usage bias in the ETEC F18 genome. Such information further elucidates the mechanisms of synonymous codon usage bias and provides the basis of molecular genetic engineering and evolutionary studies.     

Recent advances in the identification of the host factors involved in dengue virus replication

Dengue virus (DENV) belongs to the genus Flavivirus of the family Flaviviridae and it is primarily transmitted via Aedes aegypti and Aedes albopictus mosquitoes. The life cycle of DENV includes attachment, endocytosis, protein translation, RNA synthesis, assembly, egress, and maturation. Recent researches have indicated that a variety of host factors, including cellular proteins and microRNAs, positively or negatively regulate the DENV replication process. This review summarizes the latest findings (from 2014 to 2016) in the identification of the host factors involved in the DENV life cycle and Dengue infection.

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Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model

Dengue virus (DENV) causes dengue fever and severe hemorrhagic fever in humans and is primarily transmitted by Aedes aegypti and A. albopictus mosquitoes. The incidence of DENV infection has been gradually increasing in recent years due to global urbanization and international travel. Understanding the virulence determinants in host and vector transmissibility of emerging epidemic DENV will be critical to combat potential outbreaks. The DENV serotype 2 (DENV-2), which caused a widespread outbreak in Taiwan in 2015 (TW2015), is of the Cosmopolitan genotype and is phylogenetically related to the virus strain linked to another large outbreak in Indonesia in 2015. We found that the TW2015 virus was highly virulent in type I and type II interferon-deficient mice, with robust replication in spleen, lung, and intestine. The TW2015 virus also had high transmissibility to Aedes mosquitoes and could be effectively spread in a continuous mosquitoes-mouse-mosquitoes-mouse transmission cycle. By making 16681-based mutants carrying different segments of the TW2015 virus, we identified the structural pre-membrane (prM) and envelope (E) genes as key virulence determinants in the host, with involvement in the high transmissibility of the TW2015 virus in mosquitoes. The transmission mouse model will make a useful platform for evaluation of DENV with high epidemic potential and development of new strategies against dengue outbreaks.     

Chikungunya Virus and Aedes Mosquitoes: Saliva Is Infectious as soon as Two Days after Oral Infection

Background

Aedes aegypti and Aedes albopictus are potential vectors of chikungunya virus (CHIKV). The recent CHIKV outbreaks were caused by a new variant characterized by a mutation in the E1 glycoprotein gene (E1-226V) which has favored a better transmissibility by Ae. albopictus. As Ae. albopictus tends to replace Ae. aegypti in many regions, one question remained: is Ae. albopictus as efficient as Ae. aegypti to transmit the variant E1-226V of CHIKV?

Methodology and Findings

We infected orally both species with the variant E1-226V and estimated the infection, the viral dissemination, and the transmission rate by real time RT-PCR. Additionally, we used an in vitro assay to determine the amount of virus delivered by mosquitoes in their saliva. We found that Ae. aegypti as well as Ae. albopictus ensured a high replication of the virus which underwent an efficient dissemination as detectable in the salivary glands at day 2 post-infection (pi). Infectious CHIKV particles were delivered by salivary glands from day 2 with a maximum at day 6 pi for Ae. albopictus (10^3.3 PFU) and day 7 pi for Ae. aegypti (10^2.5 PFU).

Conclusions

Ae. albopictus is slightly more efficient than Ae. aegypti to transmit the variant E1-226V of CHIKV. These results will help to design an efficient vector control to limit transmission as soon as the first human cases are diagnosed.     

Chikungunya Virus Transmission Potential by Local Aedes Mosquitoes in the Americas and Europe

Background

Chikungunya virus (CHIKV), mainly transmitted in urban areas by the mosquitoes Aedes aegypti and Aedes albopictus, constitutes a major public health problem. In late 2013, CHIKV emerged on Saint-Martin Island in the Caribbean and spread throughout the region reaching more than 40 countries. Thus far, Ae. aegypti mosquitoes have been implicated as the sole vector in the outbreaks, leading to the hypothesis that CHIKV spread could be limited only to regions where this mosquito species is dominant.

Methodology/Principal Findings

We determined the ability of local populations of Ae. aegypti and Ae. albopictus from the Americas and Europe to transmit the CHIKV strain of the Asian genotype isolated from Saint-Martin Island (CHIKV_SM) during the recent epidemic, and an East-Central-South African (ECSA) genotype CHIKV strain isolated from La Réunion Island (CHIKV_LR) as a well-characterized control virus. We also evaluated the effect of temperature on transmission of CHIKV_SM by European Ae. albopictus. We found that (i) Aedes aegypti from Saint-Martin Island transmit CHIKV_SM and CHIKV_LR with similar efficiency, (ii) Ae. aegypti from the Americas display similar transmission efficiency for CHIKV_SM, (iii) American and European populations of the alternative vector species Ae. albopictus were as competent as Ae. aegypti populations with respect to transmission of CHIKV_SM and (iv) exposure of European Ae. albopictus to low temperatures (20°C) significantly reduced the transmission potential for CHIKV_SM.

Conclusions/Significance

CHIKV strains belonging to the ECSA genotype could also have initiated local transmission in the new world. Additionally, the ongoing CHIKV outbreak in the Americas could potentially spread throughout Ae. aegypti- and Ae. albopictus-infested regions of the Americas with possible imported cases of CHIKV to Ae. albopictus-infested regions in Europe. Colder temperatures may decrease the local transmission of CHIKV_SM by European Ae. albopictus, potentially explaining the lack of autochthonous transmission of CHIKV_SM in Europe despite the hundreds of imported CHIKV cases returning from the Caribbean.     

Satyrization without evidence of successful insemination from interspecific mating between invasive mosquitoes

Previous research has documented low frequencies of interspecific mating in nature between the invasive vectors Aedes aegypti and Aedes albopictus. It is also known that heterospecific male accessory gland substances transferred during mating sterilize A. aegypti but not A. albopictus females, leading to satyrization, a form of reproductive interference. This paper demonstrates that satyrization of A. aegypti by A. albopictus may occur without evidence of successful insemination. Our results show that A. aegypti females, previously exposed to A. albopictus males, are rendered refractory to subsequent conspecific mating even though their spermathecae contain no heterospecific sperm. Additional experiments demonstrating transfer of labelled semen from A. albopictus males to A. aegypti females and low production of viable eggs of females housed with conspecific males, following exposure to A. albopictus males, confirm higher incidences of satyrization than expected, based on heterospecific insemination rates. We conclude that frequencies of satyrization based on detection of interspecific sperm in spermathecae may underestimate the impact of this form of reproductive interference.     

The tendency of lentiviral open reading frames to become A-rich: constraints imposed by viral genome organization and cellular tRNA availability

Human immunodeficiency virus type 1 (HIV-1) and other lentiviridae demonstrate a strong preference for the A-nucleotide, which can account for up to 40% of the viral RNA genome. The biological mechanism responsible for this nucleotide bias is currently unknown. The increased A-content of these viral genomes corresponds to the typical use of synonymous codons by all members of the lentiviral family (HIV, SIV, BIV, FIV, CAEV, EIAV, visna) and the human spuma retrovirus, but not by other retroviruses like the human T-cell leukemia viruses HTLV-I and HTLV-II. In this article, we analyzed A-bias for all codon groups in all open reading frames of several lentiviruses. The extent of lentiviral codon bias could be related to host cellular translation. By calculating codon bias indices (CBIs), we were able to demonstrate an inverse correlation between the extent of codon bias and the rate of translation of individual reading frames in these viruses. Specifically, the shift toward A-rich codons is more pronounced in pol than in gag lentiviral genes. Since it is known that Gag synthesis exceeds Pol synthesis by a factor of 20 due to infrequent ribosomal frame-shifting during translation of the gap-pol mRNA molecule, we propose that the aminoacyl-tRNA availability in the host cell restricts the lentiviral preference for A-rich codons. In addition, less A-nucleotides were found in regions of the viral genome encoding multiple functions; e.g., overlapping reading frames (tat-rev-env) or in genes that overlap regulatory sequences (nef-LTR region). Finally, the characteristics of lentiviral codon usage are presented as a phylogenetic tree without the need for prior sequence alignment.Correspondence to: B. Berkhout     

Differential Susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika Virus

BackgroundSince the major outbreak in 2007 in the Yap Island, Zika virus (ZIKV) causing dengue-like syndromes has affected multiple islands of the South Pacific region. In May 2015, the virus was detected in Brazil and then spread through South and Central America. In December 2015, ZIKV was detected in French Guiana and Martinique. The aim of the study was to evaluate the vector competence of the mosquito spp. Aedes aegypti and Aedes albopictus from the Caribbean (Martinique, Guadeloupe), North America (southern United States), South America (Brazil, French Guiana) for the currently circulating Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia.Conclusions/SignificanceThis study suggests that although susceptible to infection, Ae. aegypti and Ae. albopictus were unexpectedly low competent vectors for ZIKV. This may suggest that other factors such as the large naïve population for ZIKV and the high densities of human-biting mosquitoes contribute to the rapid spread of ZIKV during the current outbreak.