MicrobiologyBytes

Arthropod-borne viruses (arboviruses) are transmitted biologically among vertebrate hosts by hematophagous (blood feeding) arthropod vectors such as mosquitoes and other biting flies, and ticks. Being, by definition, biologically transmitted, arboviruses must replicate in the arthropod vector prior to transmission, as opposed to being mechanically transmitted, without replication in the vector, through contaminated mouthparts. Biological transmission can be vertical, involving the passage of the virus from an infected female vector to both male and female offspring. Horizontal transmission can be venereal, from a vertically infected male directly to a female vector, as well as oral from a female vector to a vertebrate host via the saliva during blood feeding. The latter horizontal mode of transmission is most common for the majority of arboviruses and involves infection of the vector alimentary tract following a viremic bloodmeal, dissemination of the virus in the vector, and eventual virus replication in the salivary glands, followed by the injection of infectious saliva during blood feeding.

The arboviruses include a wide variety of RNA virus taxa including the alphaviruses (genus Alphavirus, one of two genera in the family Togaviridae); the flaviviruses (genus Flavivirus, one of three genera in the family Flaviviridae); the bunyaviruses (Bunyaviridae: Bunyavirus), nairoviruses (Bunyaviridae: Nairovirus) and phleboviruses (Bunyaviridae: Phlebovirus); the orbiviruses (one of nine genera in the family Reoviridae); the vesiculoviruses (one of six genera in the family Rhabdoviridae) and the thogotoviruses (one of four genera in the family Orthomyxoviridae). These groups of RNA viruses have a variety of types of RNA genomes and replication strategies, suggesting that the arthropod-borne transmission strategy has arisen many times during the evolution of RNA viruses. The only known DNA arbovirus is African swine fever virus (Asfarviridae: Asfarvirus), and the paucity of DNA arboviruses suggests that the greater genetic plasticity and higher mutation rates exhibited by RNA viruses allow them to accommodate a cycle of alternating replication in disparate vertebrate and invertebrate hosts.

Arboviruses circulate among wild animals, and cause disease after spillover transmission to humans and/or domestic animals that are incidental or dead-end hosts. Viruses such as dengue (DENV) and chikungunya (CHIKV) that have lost the requirement for enzootic amplification now produce extensive epidemics. Many arboviruses that have evolved and diversified in the tropics have produced virulent and invasive strains that have caused major outbreaks at temperate latitudes. The ability of these viruses to cause human disease depends on factors ranging from epidemiology to viral genetics. Herein, we review how some of these factors have led to arboviral emergences and resulted in human disease, by using several examples of viruses with a known epidemic history as well as some that have a poorly recognized epidemic potential.

Present and future arboviral threats. Antiviral Res. 2010 85(2): 328-345
Arthropod-borne viruses (arboviruses) are important causes of human disease nearly worldwide. All arboviruses circulate among wild animals, and many cause disease after spillover transmission to humans and agriculturally important domestic animals that are incidental or dead-end hosts. Viruses such as dengue (DENV) and chikungunya (CHIKV) that have lost the requirement for enzootic amplification now produce extensive epidemics in tropical urban centers. Many arboviruses recently have increased in importance as human and veterinary pathogens using a variety of mechanisms. Beginning in 1999, West Nile virus (WNV) underwent a dramatic geographic expansion into the Americas. High amplification associated with avian virulence coupled with adaptation for replication at higher temperatures in mosquito vectors, has caused the largest epidemic of arboviral encephalitis ever reported in the Americas. Japanese encephalitis virus (JEV), the most frequent arboviral cause of encephalitis worldwide, has spread throughout most of Asia and as far south as Australia from its putative origin in Indonesia and Malaysia. JEV has caused major epidemics as it invaded new areas, often enabled by rice culture and amplification in domesticated swine. Rift Valley fever virus (RVFV), another arbovirus that infects humans after amplification in domesticated animals, undergoes epizootic transmission during wet years following droughts. Warming of the Indian Ocean, linked to the El Niño-Southern Oscillation in the Pacific, leads to heavy rainfall in east Africa inundating surface pools and vertically infected mosquito eggs laid during previous seasons. Like WNV, JEV and RVFV could become epizootic and epidemic in the Americas if introduced unintentionally via commerce or intentionally for nefarious purposes. Climate warming also could facilitate the expansion of the distributions of many arboviruses, as documented for bluetongue viruses (BTV), major pathogens of ruminants. BTV, especially BTV-8, invaded Europe after climate warming and enabled the major midge vector to expand is distribution northward into southern Europe, extending the transmission season and vectorial capacity of local midge species. Perhaps the greatest health risk of arboviral emergence comes from extensive tropical urbanization and the colonization of this expanding habitat by the highly anthropophilic (attracted to humans) mosquito, Aedes aegypti. These factors led to the emergence of permanent endemic cycles of urban DENV and CHIKV, as well as seasonal interhuman transmission of yellow fever virus. The recent invasion into the Americas, Europe and Africa by Aedes albopictus, an important CHIKV and secondary DENV vector, could enhance urban transmission of these viruses in tropical as well as temperate regions. The minimal requirements for sustained endemic arbovirus transmission, adequate human viremia and vector competence of Ae. aegypti and/or Ae. albopictus, may be met by two other viruses with the potential to become major human pathogens: Venezuelan equine encephalitis virus, already an important cause of neurological disease in humans and equids throughout the Americas, and Mayaro virus, a close relative of CHIKV that produces a comparably debilitating arthralgic disease in South America. Further research is needed to understand the potential of these and other arboviruses to emerge in the future, invade new geographic areas, and become important public and veterinary health problems.

Lentiviruses owe their name (lenti means slow in latin) to the long period of time elapsing between the initial infection and the onset of the disease, that can protract over a period of months or even years. Viruses belonging to the Lentivirus genus are present in primates, ungulates (horse, cattle, sheep and goat) and felids (cat). Primates are the natural host for several lineages of closely related simian and human immunodeficiency viruses (SIV and HIV) that are the causative agents of acquired immunodeficiency syndrome (AIDS).

Lentivirus vectors bears an obvious advantage over other retrovirus vectors in that they offer the possibility to efficiently target non-dividing and differentiated cells, such as neurons. Paradoxically, the use of retrovirus vectors is hindered by the same process that makes them interesting for gene therapy, i.e., integration. This process is largely nonspecific and, as it has been shown in vivo, may either be of no consequence to the cell or lead to serious drawbacks. Although this problem may in theory be minimized in gene therapy applications targeting terminally differentiated cells, the problem of integration is serious. To this end, a number of alternative strategies have been developed, ranging from the redirection of retrovirus integration to particular chromosomal locations, to the ablation of the integration process altogether. Although in its infancy, the efforts to redirect retrovirus integration must be pursued and researchers may possibly transpose to lentiviruses a mechanism of specific integration used by other viruses.

The Inside Out of Lentiviral Vectors. (2011) Viruses 3(2): 132-159; doi:10.3390/v3020132
Lentiviruses induce a wide variety of pathologies in different animal species. A common feature of the replicative cycle of these viruses is their ability to target non-dividing cells, a property that constitutes an extremely attractive asset in gene therapy. In this review, we shall describe the main basic aspects of the virology of lentiviruses that were exploited to obtain efficient gene transfer vectors. In addition, we shall discuss some of the hurdles that oppose the efficient genetic modification mediated by lentiviral vectors and the strategies that are being developed to circumvent them.

Related:

• Retroviruses
• Genomic fossils in lemurs shed light on HIV
• Why is HIV a pathogen?

The fact that all other viruses encapsidate single copy genomes begs the question of why retroviruses co-package two genomics RNSa (gRNAs). One reason may be economy of scale: RNA dimerization allows formation of a unique structure – the dimer linkage – that distinguishes gRNAs from mRNAs. Monomeric HIV-1 RNAs are packaged when engineered with tandem dimer linkages, underscoring the importance of this RNA structure, and not gRNA counting per se, to packaging.

Co-packaging gRNAs allows retroviruses to generate intact proviruses despite pervasive gRNA nicking. Template switching during reverse transcription is probably why retroviruses maintain infectivity when their gRNAs are damaged by gamma rays, and why retroviruses are much less radiation-sensitive than RNA viruses like vescicular stomatitis virus. Researchers previously thought retroviral recombination might be mutagenic, but these notions have been dispelled. HIV-1 particles with two gRNAs generate full-length proviruses more efficiently than virions engineered to contain single gRNAs. Thus, another advantage of gRNA dimers appears to be increased replication fidelity.

Co-packaging gRNAs promotes higher recombination frequencies for retroviruses than all other viruses, allowing rapid loss of deleterious alleles and re-assortment of genome segments. With approximately three to ten crossovers occurring during the synthesis of every provirus, recombination is perhaps 10-fold more frequent than reverse transcriptase base substitution rates, and is an evolutionary driving force for retroviruses such as HIV-1 that display high levels of replication and multi-strain infection.

These observations help seal the case for likely evolutionary advantages of dimeric genome packaging. The DLS in its immature form, which results only upon association of two gRNAs, likely provides the means for selective gRNA packaging. The need to generate an intact provirus provides a strong motive for packaging redundant genetic information. And because retroviruses encapsidate gRNA dimers, recombination can provide the opportunity for almost limitless combinatorial genetic sampling.

Retroviral RNA Dimerization and Packaging: The What, How, When, Where, and Why. (2010) PLoS Pathog 6(10): e1001007. doi:10.1371/journal.ppat.1001007

Related:

• HIV-1 Genomic RNA Trafficking
• The shape of HIV RNA

Pneumocystis species are ascomycetous fungi that obligatorily dwell with no apparent ill effect in the lungs of normal mammals, but they become pathogenic when host defenses are compromised. Identified more than 100 years ago, these atypical fungi manifest characteristics that are unique within the Fungi, such as the lack of ergosterol, genetic complexity of surface antigens, and antigenic variation. Thought to be confined to the severely immunocompromised host, Pneumocystis spp. are being associated with new population niches owing to the advent of immunomodulatory therapies and increased numbers of patients suffering from chronic diseases. The inability to grow Pneumocystis spp. outside the mammalian lung has thwarted progress toward understanding their basic biology, but via the use of new genetic tools and other strategies, researchers are beginning to uncover their biological and genetic characteristics including a biphasic life cycle, significant metabolic capacities, and modulation of lifestyles. This review describes the alternative lifestyles indulged in by these organisms.

Stealth and opportunism: alternative lifestyles of species in the fungal genus Pneumocystis. Annu Rev Microbiol. 2010 64: 431-452

Related:

• Fungal Infections

Trypanosomes are single-celled parasites that cause sleeping sickness in humans and wasting diseases in livestock. They are transmitted by the tsetse fly and, until now, it was unclear whether they reproduce sexually or asexually, because this stage in their life cycle occurs inside the insect carrier. Sexual reproduction produces offspring that inherit half their genetic material from each parent. The alternative is asexual reproduction, where the offspring inherit all genetic material from a single parent. Sexual reproduction is important in organisms that cause diseases because it can spread genes that make them more virulent, or resistant to drugs used for treatment, as well as creating completely new strains with combinations of genes not previously encountered. Some time ago it was shown that genetic shuffling could occur when two different trypanosome strains were mixed in the tsetse fly, but it was far from clear that this was true sexual reproduction. It was difficult to visualise the process directly because it happened inside the insect. To get round this problem, Professor Wendy Gibson and colleagues used fluorescently tagged proteins to make trypanosomes light up like tiny lightbulbs. They tagged proteins that function only during meiosis, the process of cellular division at the core of sexual reproduction that shuffles the parental genes and deals them out in new combinations to the offspring.

Read more: Caught in the act: trypanosome sex visualised for the first time

Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly. PNAS USA February 14 2011 doi: 10.1073/pnas.1019423108

BK virus (BKV) and JC virus (JCV) are widespread human polyomaviruses, and their pathogenic potential during immunodeficiency has been clearly documented. Primary infection usually occurs asymptomatically (or with only mild respiratory symptoms) during childhood, after which the polyomaviruses persist latently in various organs, mainly in the urogenital system, brain and circulating leukocytes. Reactivation of both viruses is common, and frequently associated with asymptomatic viruria. The natural history of infection is well established, but it is still not clear how BKV and JCV are transmitted, although the hypotheses include respiratory, oral-fecal and urinary transmission. Furthermore, on the basis of the frequency of Polyomavirus (PV) infection in childhood, and as has been previously demonstrated in the case of animal homologue polyomaviruses such as the Murine and Simian polyomaviruses (SV-40), some authors have investigated the possibility of vertical transmission, but with conflicting results. So how are these pathogenic viruses transmitted between humans?

Serologic evidence of vertical transmission of JC and BK polyomaviruses in humans. J Gen Virol. Feb 9 2011
Vertical transmission of JC virus and BK virus has been investigated by few authors, with conflicting results. We performed a combined serological and genomic study of 19 unselected pregnant women and their newborns. Blood and urine samples were collected during each gestational trimester in the pregnant women; umbilical cord blood, peripheral blood, urine, and nasopharyngeal secretion samples were taken from newborns at delivery, one week and one month of life. Polyomavirus DNA was detected by nested-PCR. Polyomavirus IgG, IgM and IgA specific antibodies were measured in maternal and newborn serum samples using virus-like particle-based ELISA method. BKV and JCV DNA was detected in urine from 4 (21%) and 5 (26%) women, respectively. BKV and JCV seroprevalence in the pregnant women was 84% and 42%, respectively. Using a rise in the IgG level or the transient appearance of an IgA or IgM response as evidence of infection in the newborn we detected BKV and JCV infections in four (21%) and three (16%) newborns respectively. Three infants had serological evidence of infection with both BKV and JCV. In two of the four possible BKV infected newborns, the mothers seroconverted during pregnancy, while another mother was viruric and IgA seropositive. The mother of one of three possible JCV infected newborn was viruric and IgA seropositive, another mother was viruric. These results suggest JC virus and BK virus can be transmitted from mother to newborn during pregnancy or soon after birth.

Related:

• Solving the mystery of BK virus transmission
• Polyomavirus and Human Merkel Cell Carcinoma
• High prevalence of infection with three new human polyomaviruses

One of the most significant recent advances in biomedical research has been the discovery of the approximately 22-nt-long class of noncoding RNAs designated microRNAs (miRNAs). These regulatory RNAs provide a unique level of post-transcriptional gene regulation that modulates a range of fundamental cellular processes. Several viruses, especially herpesviruses, also encode miRNAs, and over 200 viral miRNAs have now been identified. Current evidence indicates that viruses use these miRNAs to manipulate both cellular and viral gene expression. Furthermore, viral infection can exert a profound impact on the cellular miRNA expression profile, and several RNA viruses have been reported to interact directly with cellular miRNAs and/or to use these miRNAs to augment their replication potential. This review article discusses our current knowledge of viral miRNAs and virally influenced cellular miRNAs and their relationship to viral infection.

Viruses, microRNAs, and host interactions. Annu Rev Microbiol. 2010 64: 123-141

Related:

• Five Questions about Viruses and MicroRNAs
• It’s not the size of your RNA, it’s what you do with it that counts
• Virus-encoded microRNAs in herpesvirus biology
• MicroRNAs in Picornavirus Infection

New research is beginning to crack the problem of which strain of flu will be prevalent in a given year, with major implications for global public health preparedness. A computational study of 40 years of flu genomes offers a new way of looking at mutations: by cataloging pairs of genetic changes that have occurred in rapid succession, observing that a mutation in one half of the pair can act as an early warning sign of a mutation about to occur in the other.

Tracking single mutations in a vacuum is not always enough to understand how the flu virus evolves. Sometimes a mutation is functional or adaptive only if it’s in the context of a certain genetic background – that is, if the protein already has some other mutation. The influence such combinations have on an organism’s adaptive fitness is known as epistasis. If you see a mutation occur in Site A and then very soon after you see a mutation in Site B, and this pattern happens repeatedly, then you have some evidence that A and B influence fitness epistatically. The first mutation might be useless on its own, but it might be a prerequisite for the second mutation to be useful. The first mutation is like giving you a nail, and the second one is like giving you a hammer.

Because the studied mutations generally affect the surface proteins that determine whether the virus can enter and infect human cells, being able to predict what mutations are likely to happen in the near future has lifesaving applications. Tens of thousands of Americans, and hundreds of thousands worldwide, die of seasonal flu complications every year. Flu vaccine production is labor intensive and time consuming; to have enough supplies ready for the flu season, public health groups like the Centers for Disease Control and the World Health Organization must make an educated guess as to which strain is likely to be the most active several months in advance. Observing the leading site of an epistatic pair could give them a head start.

Prevalence of Epistasis in the Evolution of Influenza A Surface Proteins. (2011) PLoS Genet 7(2): e1001301. doi:10.1371/journal.pgen.1001301
The surface proteins of human influenza A viruses experience positive selection to escape both human immunity and, more recently, antiviral drug treatments. In bacteria and viruses, immune-escape and drug-resistant phenotypes often appear through a combination of several mutations that have epistatic effects on pathogen fitness. However, the extent and structure of epistasis in influenza viral proteins have not been systematically investigated. Here, we develop a novel statistical method to detect positive epistasis between pairs of sites in a protein, based on the observed temporal patterns of sequence evolution. The method rests on the simple idea that a substitution at one site should rapidly follow a substitution at another site if the sites are positively epistatic. We apply this method to the surface proteins hemagglutinin and neuraminidase of influenza A virus subtypes H3N2 and H1N1. Compared to a non-epistatic null distribution, we detect substantial amounts of epistasis and determine the identities of putatively epistatic pairs of sites. In particular, using sequence data alone, our method identifies epistatic interactions between specific sites in neuraminidase that have recently been demonstrated, in vitro, to confer resistance to the drug oseltamivir; these epistatic interactions are responsible for widespread drug resistance among H1N1 viruses circulating today. This experimental validation demonstrates the predictive power of our method to identify epistatic sites of importance for viral adaptation and public health. We conclude that epistasis plays a large role in shaping the molecular evolution of influenza viruses. In particular, sites with dN=dSv1, which would normally not be identified as positively selected, can facilitate viral adaptation through epistatic interactions with their partner sites. The knowledge of specific interactions among sites in influenza proteins may help us to predict the course of antigenic evolution and, consequently, to select more appropriate vaccines and drugs.

Related:

• Spotting pandemic influenza viruses
• How can vaccines against influenza and other virus diseases be made more effective?
• Protective immunity against influenza

“It’s hard to visualise what something as small and complex as the HIV virus actually looks like. But now Ivan Konstantinov and his team from Visual Science have created the most-detailed 3D model of the virus to date (see video above). An image of this visualisation just won first place in the 2010 International Science and Engineering Visualization Challenge, sponsored jointly by the journal Science and the National Science Foundation (NSF).  The model contains 17 different viral and cellular proteins and the membrane incorporates 160 thousand lipid molecules, of 8 different types, in the same proportions as in an actual HIV particle. It denotes the parts encoded by the virus’s own genome in orange, while grey shades indicate structures taken into the virus when it interacts with a human cell. To create the visualisation, the team consulted over 100 articles on HIV from leading science journals and talked to experts in the field. Then they reconstructed viral proteins from X-rays before assembling the structure of an entire HIV particle. The final appearance was achieved by experienced designers and 3D graphics specialists.”

Read more: New Scientist TV: HIV as you’ve never seen it before