Dozens of Cruises Depart Rio De Janeiro to Miami

Including Recife, the Coast of Brazil, and Amazon River

Zika's Path to Florida From About 30 People Began Spring 2016 Vast Majority via Cruise Ship
Credit: Google Maps World Ports [Fair Use] Cruise ship by CoolChris99 [Public domain] Text and ship route (in red) by RoseWrites

Scientists Traced Zika's Path From Brazil to the U.S.

On March 4th, 2017, a Medscape post by Damian McNamara called Genomics Reveal Surprises About Florida Zika Outbreak[1] caught my attention. The gist:

It wasn't a single or even a few people that started the outbreak in Florida, but about 30 people.

And genomic sequencing of the virus from mosquitoes and patients also revealed that Caribbean travelers were the primary means of introduction.

"The vast majority [of infected travelers] came by cruise ship. That is not something I would have expected," said Kristian Andersen, PhD, from Scripps Translational Science Institute in La Jolla, California.

Also, the Zika virus arrived in Florida much earlier than the researchers expected: sometime in the spring of 2016.





This Supports Culex Mosquitoes as Zika Vectors

Culex Breed in Open Bodies of Water, Even Polluted Water

Cruise ships often travel from Miami to Rio de Janeiro, taking port along the way. Many cruises include a tour of the Amazon river. 

These areas are definitely where more Culex and birds (as reservoir hosts) would be found. And as I mentioned before, red-whiskered bulbuls were introduced (and appear to have become established) in Rio de Janeiro in 2006.

Since West Nile virus is closely related to the Zika virus, I suspect many other species of birds (not just bulbuls or greenbuls) could be reservoir hosts too. Over 300 species of birds carry West Nile virus.



Birds, Lizards, and Frogs Take Up Temporary Housing

In "Central Park" on the Allure of the Seas Cruise Ship

Allure of the Seas "Central Park"
Credit: DaMongMan on flickr (CC-by-2.0) "Central Park" - Allure of the Seas

Do Any Cruise Ships Have Birds, I Wondered

Just One Bird Could Infect 100s of Mosquitoes With Zika

Although I cannot find any source that states which cruise ships might have carried the infected passengers, I wondered if any ships have birds (perhaps as mascots).

To my surprise, Royal Caribbean has a Central Park on their Oasis-class ships. According to Thomas Brownlee, officer in charge of Horticulture and Integrated Pest Management (IPM) for Allure of the Seas and Oasis of the Seas:

"There are also small birds, lizards, and frogs that take up temporary housing in Central Park which are all considered bio-control as they naturally help me out with little plant insects."[2]

Culex bite "small birds, lizards, and frogs".[3] In fact, some species of Culex feed exclusively on ectotherms (Woke, 1937a).

I can't help but wonder if any of these birds might be reservoir (or amplifying) hosts of the Zika virus. I will send this article to Royal Caribbean.

Remember, a lesser-known (and ignored) study by Okia et al., 1971 found that 15 percent of birds in Uganda had Zika. And almost half of little greenbuls (Eurillas virens) had Zika.


"Central Park" on a Cruise Ship Sure Looks Inviting

Yet, Culex and Aedes Could Be Present on Any Cruise Ship

Central Park of Allure of the Seas
Credit: Wilson Hui on flickr (CC-by-2.0) "Central Park" of Allure of the Seas

Zika Virus Spread Throughout the Americas

Samples Collected in Each of the Colored Countries or Territories

 Zika virus spread throughout the Americas [Source: Genome sequencing reveals Zika virus diversity and spread in the Americas]
Credit: Made available under a CC-BY-NC-ND 4.0 International license

Delving Into the Study by Metsky et al.

Genome Sequencing Reveals Zika Virus Diversity and Spread in the Americas

Posted February 18th, 2017 in BioRxiv, 61 study authors were listed in this 27-page PDF.[4] And I took my time reading it over. Key points:

  • ZIKV [Zika virus] genomes from Colombia, Honduras, and Puerto Rico each clustered into distinct clades. 
  • The study authors "observed tight clustering among ZIKV from the continental US, the Dominican Republic, and Jamaica".
  • ZIKV genomes from Brazil, Colombia, and Puerto Rico were similar and distinct from genomes sampled in other countries.
  • ZIKV from Honduras formed a third cluster that contains genomes from Guatemala or El Salvador.
The researchers observed four positions with nonsynonymous mutations that occurred on two or more branches of the phylogenetic tree; two of these (at 4287 and 8991) occurred together and might represent incorrect placement of a Brazilian branch.
They stated, the remaining two are more likely to represent multiple nonsynonymous mutations; one (at 9240) appears to involve nonsynonymous mutations to two different alleles.
So, the researchers looked for evidence of selection on the Zika virus genome.
They "searched for an excess of nonsynonymous mutations in the envelope (E) protein coding region; positive selection due to host antibody binding to this ZIKV surface protein could drive nonsynonymous changes there, as seen in other viral glycoproteins".
However, they reported "nonsynonymous substitution rate in E proved to be similar to that in the rest of the coding region; moreover, amino acid changes were significantly more conservative in the region than elsewhere".
And the study authors added:
"Any diversifying selection that is occurring in the surface protein appears to be operating under substantial selective constraint. In the ZIKV 3' UTR, we also found evidence for purifying selection; this confirms the functional importance of the region, which has previously been reported to play a role in viral replication."

Wolbachia + Zika as a Bacteriophage

Inhibits Amino Acid Changes & Increases Substitution

Wolbachia and Zika Working in Tandem; Zika a Bacteriophage
Credit: Created by RoseWrites. All Rights Reserved. Phage via NIH website; Zika virus cryo-EM by Starless [Both Public Domain]

Sites of Wolbachia-Infected Mosquito Releases

Three Close to Caribbean: Brazil, Columbia, and Florida

World Map: Wolbachia, Bulbuls, Culex as Zika Vectors, Culex in Brazil Have 2 Mitochondria
Credit: World Map by Christopher Schnese on flickr (CC-by-2.0); Bulbul by myjkccd (Own work) [Public domain] - image flipped for native regions. Concept, text, colors, Culex mosquitoes by RoseWrites.

Filling in the Gaps: Is Wolbachia Playing a Role?

The study Mosaic Nature of the Wolbachia Surface Protein[5] (wsp) by Laura Baldo, Nathan Lo, and John H. Werren states:

"... it is unclear whether selection for amino acid changes is a result of adaptation to new host environments, of antagonistic coevolution between the host and wsp HVRs, or of some combination of these two processes. Overall, both recombination and diversifying selection appear to be responsible for the extensive divergence among wsp sequences."

The outer membrane protein of Wolbachia, also known as Wolbachia surface protein (wsp), is thought to be mosaic in nature.

In the study Comparative analysis of Wolbachia surface protein in D. melanoagster, A. tabida, and B. malayi[6] by Jayaramaiah Uday and Hosagavi Puttegowda Puttaraju states:

The predicted complex structure were observed in PROCHECK 3D-validation shows 84.15% of steriochemical rotation of torsion angles and no amino acid changes in protein complex.
In WSP, there is less recombination impact and divergence of amino acids distribution in D. melanogaster and A. tabida but more diverged in B. malayi.
"The initial work on Wolbachia surface protein has shown that it may function to inhibit apoptosis of host, antigenic role and it shows strong immune response against humans who are infected with filariasis."
"We predict the structure of Wolbachia surface protein from three different sources which behaves as parasitic in D. melanogaster, mutalistic in B. malayi, both parasitic and mutalistic in A. tabida."

Higher Frequency of C-to-T and T-to-C Substitutions

Metsky et al. made mention of "a significantly higher frequency of C-to-T and T-to-C substitutions than other transitions".[4]

And they surmised that "many processes are possible contributors to this conspicuous mutation pattern, including mutational bias of the ZIKV RNA-dependent RNA polymerase, host RNA editing enzymes (e.g., APOBECs, ADARs) acting upon viral RNA, and chemical deamination, but further work is required to determine the actual cause of this phenomenon".[4]

According to the study Decreased Diversity but Increased Substitution Rate in Host mtDNA as a Consequence of Wolbachia Endosymbiont Infection[7] by D. DeWayne Shoemaker, Kelly A. Dyer, Mike Ahrens, Kevin McAbee and John Jaenike:

"Wolbachia, and probably other maternally transmitted endosymbionts, can severely depress levels of mtDNA diversity within an infected host species. In contrast, such infections may increase the rate of substitution in mtDNA."[7]
The study Asymmetrical Reinforcement and Wolbachia Infection in Drosophila[8] by John Jaenike, Kelly A. Dyer, Chad Cornish, and Miranda S Minhas mentioned "differing only in a synonymous C-to-T substitution ..."

Transient Transinfection of Wolbachia in Adult Mosquitoes Can Actually Lead to the Enhancement of Virus Infection

The 2015 study by Karyn N. Johnson called The Impact of Wolbachia on Virus Infection in Mosquitoes[9] clearly states:

"Transient transinfection of Wolbachia into adult mosquitoes can lead to enhancement of virus infection. WNV [West Nile virus] infection rate was enhanced following transient transinfection of wAlbB into Culex tarsalis mosquitoes."

And she added:

"The possibility of arbovirus enhancement is an important consideration given there is one example of a natural Wolbachia infection stimulating increased susceptibility to a DNA virus in the African armyworm and other examples of Wolbachia-induced enhancement of plasmodium infection in mosquitoes."

I would add that in my article Zika: The Warnings About Wolbachia and Culex Our Health Authorities are Ignoring, I wrote about Dr. Jason Rasgon, of Penn State’s entomology department. He investigated whether Wolbachia could help control the spread of West Nile Virus (WNV) in Culex. He expected Wolbachia to block WNV, but it didn't. 

"We had to repeat it a couple times before we actually believed the result. I can’t believe this is just a fluke. If you keep looking, you’ll probably find more examples of it." ~ Dr. Jason Rasgon

Wolbachia Doesn't Work in Natural Environments and as Time Progresses

In contrast to stably transinfected mosquitoes, those naturally infected with Wolbachia do not ubiquitously exhibit antiviral effects. Specifically, Karyn Johnson's study stated:

"Aedes albopictus mosquitoes naturally co-infected with wAlbA and wAlbB have similar total CHIKV or DENV loads to Wolbachia-free mosquitoes."

And further along, she surmised:

"... while presence of Wolbachia in Culex quinquefasciatus can lead to reduced vector competence, the Wolbachia density in natural Culex populations may not be high enough to support these antiviral effects.

As a consequence, Wolbachia may not impact vector competence in the field.

Taken together, these studies suggest that Wolbachia may not have a major impact on competence of mosquitoes with a naturally occurring Wolbachia infection to transmit arboviruses ..."

In time, adaptation occurs and she mentioned viruses could evolve to “escape” the antiviral mechanisms mediated by Wolbachia.

What if Just the Opposite Occurs? Wolbachia Enhances Zika

I think it is already happening. As detailed in my article More Proof: Wolbachia-Infected Mosquito Releases Might Be Causing the Most Devastating Zika Infections:

A  growing number of studies report that Wolbachia directly interferes with viruses and other pathogens inside the arthropod host. This direct effect of Wolbachia can either impede or promote the pathogen's replication and survival (Zug and Hammerstein, 2015).

In Australian Aedes aegypti mosquitoes, the wMelPop Wolbachia strain causes infected larvae to develop faster than uninfected individuals under low nutritional conditions and high larval density, whereas the opposite effect occurred when food availability was high and larval density low.

What happens in the natural environment is of paramount importance. It's rare that "food availability is high and larval density low" in the wild.

Wolbachia seems to enhance the fitness of mosquitoes once they adapt and, in time, I believe the Zika virus has found a way to benefit from Wolbachia.

Wolbachia in Aedes Mosquitoes Doesn't Disappear

It Can Live for at Least a Week After Its Host's Death Allowing It to Spread

How Wolbachia-Infected Aedes Enters the Food Chain
Credit: All Flickr images (CC-by-2.0): muffinn (top left image); Russell Neches (bottom left image); Peter Sheik (top right image); and Simon Kutcher, AFAP (bottom left image). Concept and text by me, RoseWrites, created on January 17th, 2017.

Kresge Eye Institute at the Wayne State University

Zika Virus Replicates In (and Mainly Affects) the Retina

I left a comment similar to this one on the article by Dana Afana called Link between Zika virus and blindness discovered at Wayne State:[10]

This actually fits my Wolbachia theory (Zika is working in tandem with Wolbachia, behaving like a bacteriophage) because of the following: 

"In Drosophila, the distribution of the pathogenic Wolbachia strain, wMelPop, in the nervous system of adults is temperature dependent, with increased temperature favoring the expansion of Wolbachia from the central brain to peripheral areas such as the optic lobe and retina (Strunov, Kiseleva, & Gottlieb, 2013)."[11]


Antibiotics Which Combat Bacteria Work On Zika (a Virus)

UC San Francisco researchers have identified fetal brain tissue cells that are targeted by the Zika virus and determined that azithromycin, a common antibiotic regarded as safe for use during pregnancy, can prevent the virus from infecting these cells.

Azithromycin is used to treat certain bacterial infections, such as bronchitis; pneumonia; sexually transmitted diseases (STD); and infections of the ears, lungs, sinuses, skin, throat, and reproductive organs. 

As mentioned in my article Zika Shrivels Testes, Drops Testosterone, and May Cause Infertility, azithromycin is also used sometimes to treat babesiosis (an infectious disease carried by ticks). 

Keep in mind, there are other reservoirs (birds) and other blood-sucking insects that could be acquiring and transporting Wolbachia and/or Zika to new species.

Researchers from the Perelman School of Medicine at the University of Pennsylvania found that nanchangmycin was a potent inhibitor of Zika virus entry across all cell types tested, including endothelial and placental cells.
The source described nanchangmycin as "an antimicrobial indentified in China as part of a natural medicinal products survey"[12] (which I found an odd way to describe it).
Nanchangmycin is a polyether ANTIBIOTIC with similar structure to dianemycin and is very active against a broad spectrum of harmful nematodes (which appears to strengthen my case that Zika is acting like a bacteriophage (relying on Wolbachia) and hitching a ride with it to the gonads). 
My point is: antibiotics appear to be working to block Zika (a virus). Antibiotics, obviously, are supposed to work on bacteria. So, the question is why are they working on Zika? Could it be surface proteins that Wolbachia is "sharing" with Zika?

D. Melanogaster (wMel) Can Never Be Taken Back

The Wolbachia endosymbiont from D. melanogaster (wMel) is used in Wolbachia-infected Aedes mosquito releases.

Yeah. Let that sink in for a moment.

According to Eliminate Dengue's own website FAQ:

As the bacteria is passed on from generation to generation and over time, the percentage of mosquitoes carrying Wolbachia grows until it remains high without any further releases.[13]

Our research has shown that Wolbachia can sustain itself in mosquito populations without continual reapplication, which makes this method sustainable and cost-effective in the long-term.[13] 

Species Will Die or Have Reproductive Failures

Zika Survives 5 Days in a Dead Host; Wolbachia x 1 Week

Wolbachia-Infected Aedes, Eggs, and Larvae Impact Many Ecosystems
Credit: CC-by-2.0 images: Arctic Tern by Tony Smith on flick; Aedes Larvae by NIAID; Mesocyclops by Simon Kutcher, AFAP.; Pregnant Woman & Man by Nina Matthews; Rufous-bellied thrush by Charlesjsharp [CC BY-SA 4.0]. All others Public Domain or by RoseWrites.

Proof: Conversation I Had With Eliminate Dengue

Eliminate Dengue on Facebook responded to me
Credit: Screenshot of Facebook conversation with Eliminate Dengue

More Recommended Reading About the Zika Virus

Egos: The Real Reason Scientists are Unable to Solve Zika Mysteries

Zika Virus: Our Tainted Blood Supply

An Open Letter to Dr. Margaret Chan, Director-General of WHO

On Zazzle, I have 140 products (so far) that educate and promote the prevention of the spread of Zika. Some might even make you laugh. Every item purchased will help fund Zika research. You can even customize most of the products.[14]

On Facebook, I have a devoted page called Zika: Let's Stop a Global Pandemic where you can keep up-to-date on the latest findings from the scientific community (not just mainstream media).[15]

Author's note: All of my citations have a clickable link to their source. The list is found in the bibliography at the end of this page.