Examine evolutionary bottlenecks to depict viral fitness landscapes
An evolutionary bottleneck is the event that reduces the size of a population, limiting the genetic diversity of the species. It occurs when viruses transmit from one host to another, rendering the functionally dominant populations remaining in the circulation among hosts. Molecular barcodes thus become a powerful tool, with which we can simply trace transmission paths of a population of the viruses over different hosts at a single-virus level. Influenza virus is one of the viruses that has subsisted in the human community over a long history. The most remarkable pandemics caused by influenza virus is the so-called Spanish flu, or the 1918 H1N1 pandemic, infecting ~30% of the population[112,113]. Influenza virus A is endemic in waterfowl and is usually nonpathogenic in these birds[114]until the emergence of H5N1 outbreaks in Asia[115–117]. Soon afterwards H5N1 influenza virus is known to be zoonoses, which can transmit from its natural host to humans[118–122]. To date, in addition to avian influenza virus A(H5N1), several zoonotic influenza viruses, such as avian influenza virus A(H7N9)[123,124]and A(H5N6)[125–127]as well as swine influenza virus A(H1N1)[128–131]and A(H3N2)[132,133]and its variant (H3N2v)[134]have been reported. Rapid antigenic variation that enables the viruses to escape from neutralizing antibodies[135]is frequently observed in influenza viruses, rendering vaccines not fully effective. It thus a requisite to understand which variations of the viruses could go through the pressure of selection bottlenecks in different cross-species transmission routes before developing a better strategy against influenza virus infection.
With barcoded influenza A virus in hand, Varble and colleagues examined viral transmission bottlenecks under the selection forces present in four different experimental systems, including Madin-Darby canine kidney cells, embryonated eggs, direct-contact transmission in guinea pigs and ferrets and respiratory-droplet transmission in ferrets[24]. In the context of population dynamics and evolution, the bottleneck concept is often used in reference to events that limit population size: only individuals which successfully pass through the bottleneck retrain in a population in a new environment. The authors detected that bottleneck selection occurred only when embryonated-chicken eggs were infected with barcoded viruses: 5 - 13 viral clones harboring unique barcodes were successfully amplified in each egg[24]. Intriguingly, such bottlenecks observed in individual recipient animals can be independent from viral genome sequences. Furthermore, the authors observed that viral transmission routes affect bottleneck stringency: survival of barcoded viruses transmitting via contact- and airborne infection showed a dramatic decrease compared with direct inoculation[24]. As such, the authors concluded that viruses like H5N1 have to overcome two distinct bottlenecks before the emergence of pandemic. The first one lies on the level of the viral genome sequence. Viruses acquire genetic mutations and/or reassortments to maintain replication fitness in recipient hosts[136]. Another is dependent on viral transmission routes. This barcoded influenza A virus has further been applied by Muñoz-Moreno and colleagues to reveal the evolutionary relationship between influenza NS1 genotypic variants and host tropism[137].
Zika virus is another example of zoonotic diseases that is through mosquito-borne transmission. It was first discovered in a Rhesus monkey and in Aedes africanus mosquitoes during a yellow fever study in the Zika forest of Uganda in 1947 and 1948[138,139]. Through the past fifteen years several outbreaks of Zika virus were seen, bringing this virus to the world’s attention. Even though the World Health Organization declared the end of the Zika virus epidemic in 2016[140], it still represents a highly significant and long-term problem. The virus induces a mild flu-like disease in about one in five infected individuals[141–144]; in other words, approximately 80% of Zika infections are asymptomatic. Pathologically speaking, Zika virus can also infect the placenta, transmitting from pregnant women to fetuses[145]in addition to blood-brain barrier endothelial cells, neurons and neural stem cells[146,147]. Even though the frequency of vertical transmission is not clear, current data suggest that it may be a very common case, especially if infection occurs in the period of the first trimester[148]. At least 5% of babies contaminated by Zika virus during maternal pregnancy suffered a collection of neurological, visual, auditory, and developmental birth defects, so called congenital Zika syndrome[145]. It is now also known that Zika virus infection is closely associated with diseases. In Brazil the outbreak of Zika virus correlated with an increase in cases of infant microcephaly[149,150], and in other parts of the Amercias there has been a significant increase in Guillain-Barré syndrome coinciding with Zika infections[151].
In order to better understand the in vivo replication and evolutionary dynamics of Zika virus infection, Aliota and colleagues[26]employed barcoded Zika viruses to track and monitor individual viral lineages during acute infection in pregnant and nonpregnant macaques. The authors first confirmed that viral RNA from barcoded Zika virus was detectable in placentae. Fetal histology also showed several pathological symptoms relevant to Zika virus infections[26], meaning that this animal model with barcoded Zika virus infection can most likely bring us the picture representing Zika virus infections in humans. Although no significant changes in the frequency of barcode distribution was observed in nonpregnant macaques, a bottleneck effect appeared after seven dpi in pregnant macaques[26]: the prevalence of the dominant viral lineages identified by unique barcodes changes mainly after day 8 post infection. Aliota and colleagues further used mosquitos fed with Aedes aegypti vector competence for barcoded Zika virus to infect the pregnant macaque. Only one viral lineage, Zika_BC02, representing the second most dominant barcode present in the early period of infection was successfully transmitted via mosquitoes.