A multi-year University of Florida program that monitored school-aged children in Haiti for unexplained fevers has uncovered unusual evidence of past viral circulation that went unnoticed up to now.
UF researchers recently published a paper in PLOS Neglected Tropical Diseases that reports the first known case of Melao virus in humans, and the first known detection of Oropouche virus in Haitians. The viruses were found in blood samples obtained in 2014. While they do not provide information about current community public health, the findings add insight to the dynamics of how viruses transmitted by arthropods—such as ticks and mosquitoes—move between animal and human hosts, and across geographic areas.
Melao and Oropouche viruses both belong to the same taxonomic genus, Orthobunyavirus, which is found worldwide. In North America, viruses in the Orthobunyavirus group can cause swelling in the brain, or encephalitis. In the US, familiar viruses from this group include La Cross and Jamestown Canyon viruses. These cause the second and third leading insect-borne viruses in the country that invade the nervous system.
Prior work from the UF-led program in Haiti has identified unusual occurrences of Madariaga virus, Mayaro virus, and porcine deltacoronavirus. The team includes UF researchers spanning the College of Public Health and Health Professions and the College of Medicine, and the laboratory work is performed within biosecure facilities in UF’s state-of-the-art Emerging Pathogens Institute.
“These new findings may seem a little obscure at first,” says the director of UF’s Emerging Pathogens Institute, J. Glenn Morris, Jr., M.D. “But they are important for understanding the background rate at which viruses move between animals and people, and for tracking how viruses move between South America and the Caribbean.”
The Caribbean as Florida’s sentinel
UF’s EPI has maintained an interest in infectious diseases in Haiti since the aftermath of a cholera outbreak following a devastating earthquake more than a decade ago. UF researchers worked in partnership with staff at the Christianville Foundation school in Haiti’s Gressier region between 2014 and 2019. The Christianville Foundation includes several school campuses plus a health clinic that provides care to about 1,250 children.
When a child came to the school’s clinic with a fever that could not be explained or diagnosed, the staff obtained consent to take a blood sample. This biospecimen was then securely transferred to UF’s EPI facility which has CDC-approved laboratories, equipment, and protocols in place to safely study infectious agents.
“Haiti and the Caribbean matter to us, because what is found there can easily come to mainland Florida and the rest of the U.S.,” Dr. Morris says. “It benefits everyone for us to keep an eye on what is emerging in Haiti. But it’s a two-way street. We have also found viruses that moved into there from the US.”
Most modern diagnostic virology work is done using molecular technologies because they are fast and highly sensitive. But because they are designed to look for a certain type of virus—Zika, for example—if the results are negative, they don’t allow for the discovery of the virus that is making someone ill.
Research team member John Lednicky, a professor in the College of Public Health and Health Professions, and an EPI faculty member, says: “Because they detect only one virus, the use of these tests is limited to a narrow menu, whereas there are tens of thousands of viruses that are known to or potentially affect humans. We often miss the forest for the trees when we use commonly available molecular tests to identify viruses.”
The UF team takes a different approach, one akin to molecular detective work. They use what’s referred to as an unbiased approach to both grow and then identify unknown viruses that may be present in a biospecimen.
First, they screen the biospecimen—usually it’s blood—with diagnostic tests that look for a known pathogen, such as dengue, chikungunya or Zika viruses, or malaria parasites. If these screens fail to identify an infectious agent, they next add a portion of the specimen to cell cultures and attempt to grow any viruses that may be present.
Next, if the cell cultures then show signs of infection, which appear as visibly altered or dead cells, they then use unbiased reverse transcription-polymerase chain reaction testing. These methods amplify fragments of virus genomes and then genetic sequences are used to establish the most likely identity of the virus.
Last, they use global sequencing data repositories to find a match for the viral genetic sequences in their samples. After it’s identified, they do a phylogenetic analysis; this identifies the most closely related known viral strains. This information helps to determine what strains the virus in the sample is most likely related to, and where in the world it has been found before. This gives researchers an idea of how viruses evolve and spread geographically.
By honing this unusual approach in detecting and identifying emerging viruses, the team has uncovered emerging viruses that may otherwise go undiagnosed and unnoticed.
“At its core, our process opens doors because we are not limiting ourselves to what’s already been discovered,” Dr. Morris says. “When you administer a test for Zika virus, you get a positive or negative result. But a negative result still doesn’t answer the question of what has sickened the child. What we do is find that cause.”
The efforts to grow the viruses in cell cultures and the genetic sequencing is led by John Lednicky. The phylogenetic analysis is led by Marco Salemi, a professor of experimental pathology in UF’s College of Medicine, and an EPI faculty member. And EPI’s Dr. Morris oversaw the clinical aspects of the Haiti program while also bringing his expertise in epidemiology to interpreting how the viruses may have jumped from animals to people, and whether transmission between humans occurred.
First in human, first in Haiti
The team’s latest work describes detection of Melao virus for the first time in humans. This virus was first found in mosquitoes from Trinidad and Tobago. in the 1960s. Antibodies against it have been found in both horses and people, which suggests that both can become infected. But the antibodies may also be a form of cross-reaction that indicates past infection with a virus genetically close to, but different than, Melao virus. Regardless, until now no one has documented people becoming sick with Melao virus, the virus itself had not been found in anyone, nor had it been detected in Haiti previously.
The UF researchers documented Melao virus in five Haitian school-aged children. The children attend four different campuses within the participating school’s academic units, and there was no movement of students between the campuses. This indicates that the children did not contract Melao virus from each other and that it could have been circulating within their community in late 2014.
The affected children experienced fever, abdominal pain, and swollen lymph nodes. The symptoms are too vague to identify Melao virus clinically, the researchers say. Future research should examine whether adults can have asymptomatic infections with Melao virus, and whether it can cause neuroinvasive infections as can other related orthobunyaviruses.
“It is no wonder that travelers to the Caribbean and surrounding areas develop ‘mysterious’ tropical diseases,” Lednicky says. “We have not cataloged many of the viruses in circulation and do not have tests to detect them. Our approach broadens our understanding of the pathogens circulating in Haiti, raising awareness that orthobunyaviruses occur there, and more importantly, exposes gaps in our knowledge of vector-borne viruses circulating in the Caribbean.”
Similarly, the UF researchers also found Oropouche virus in Haiti for the first time. While it is known to circulate in Brazil, its discovery in Haiti indicates that it may have a wider geographic presence than previously understood. The patient in whom the virus was found was diagnosed based on clinical symptoms as having chikungunya virus. Misdiagnosis is not uncommon with arthropod-borne viruses, according to Lednicky, because the symptoms tend to be similar: fever, rash and body aches.
“Outbreaks by these viruses can overlap or coincide,” Lednicky says. “During the recent chikungunya outbreak in Haiti, doctors mistook cases of dengue fever and Zika for chikungunya. But proper diagnoses are essential for optimal medical management, as treatments for one type of vector-borne virus may be ineffective for another type.”
A history of firsts
The UF team’s work to identify emerging viruses in Haiti is building an understanding of the dynamics of how arthropod viruses spread and their history within a region. This lets researchers know whether the virus was recently introduced; and if it was, provides guidance on interventions to control or minimize further spread.
“Often, when a virus is first detected in a new place, the importance is not always obvious,” Lednicky says. “A good example of this is Zika virus, which we found in blood samples from Haitians in 2014, before widespread reports of its significance as a human pathogen started emanating from Brazil in 2015. Before the reports from Brazil, few people paid attention to our Zika virus discovery.”
The team’s two latest discoveries follow several years of detective work in which they documented a range of viruses found for the first time in people or in the Caribbean. These are their main findings:
- Discovered the first instance of a porcine deltacoronavirus virus in a human, in Haiti.
- Detected and identified the first instance of keystone virus in a human, found in a Florida teenager.
- Discovered the first instance of Madariaga virus in the northern Caribbean, in a Haitian child.
- Discovered the first instance of Mayaro virus in a Haitian child and established genetic links between strains found in Haiti and those known from Brazil.
- Discovered a coronavirus strain in Haitian child which had newly emerged from Malaysia and presented with gastrointestinal symptoms.
- Discovered an unusual case of Madariaga virus in a Venezuelan child, which may have spilled over into humans from horses.
- Discovered a strain of non-polio enterovirus D68, which was known from New York, in Haiti and the broader Caribbean for the first time.
- Documented that Zika virus was present in Haiti in 2014 before the major outbreak of 2015 in Brazil.
- Discovered a new subgroup of Chikungunya viruses unique to America, and Spondweni virus,collected from mosquitoes in Haiti.
- Documented the range of arboviruses infecting people in Haiti as the chikungunya epidemic occurred in 2014-2015.
Dr. Morris says he has been asked why the researchers have continued working in Haiti, despite the political volatility and social unrest. While the program has experienced some disruptions in surveilling blood samples from the schoolchildren, he has continued to push for sustaining the program.
“Recent events have undoubtedly exacerbated public health risks in Haiti,” Dr. Morris says. “But we do this because there is a place like Haiti that needs our expertise and help.”