Stalking arboviruses

March 18, 2019: EPI and UF College of Veterinary Medicine researcher Maureen Long helped build a patient surveillance system in Pakistan to track and diagnose mosquito-borne diseases. The project captured outbreaks and laid the groundwork for a countrywide surveillance, detection and reporting system.

Stalking arboviruses

Maureen Long’s colorful virology career spans from horses to humans, but the patient is less the object of fascination for her than the agent that infects it. Specifically, viruses that hitch rides on a mosquito’s delicate bite.

“Every virus tells its own story,” said Long, a member of UF’s Emerging Pathogens Institute and a large animal internist and infectious disease researcher in the College of Veterinary Medicine. “Every time a disease emerges, I find it fascinating because these diseases are writing their own story right in front of you. It’s like seeing something write its own book.”

Maureen Long working with lab samples in BSL

Maureen Long, DVM PhD, extracts RNA from patient samples to test for the presence of mosquito-borne diseases, also known as arboviruses. (UF Health Creative Services)

Long’s work on Pakistan, however, is rewriting the country’s narrative of contemporary mosquito-borne diseases. Over the past six years, Long, who is also a professor of virology/microbiology, helped document the first known outbreak of West Nile Virus in Pakistan to cause neurological symptoms in humans, as well as the first known outbreak of Chikungunya in the country’s history. Future work just may assist Pakistani health professionals and officials to create a surveillance system to detect and report mosquito-borne viral diseases.

From Florida to Asia

As an infectious disease researcher specializing in large animals, Long has had plentiful fodder for study throughout her career in Florida: West Nile Virus, Eastern equine encephalitis virus, Potomac horse fever, and Neospora caninum, to name a few. But it’s the mosquito-borne viruses, known more fancifully as arboviruses, that captivate her. Arboviruses are prevalent across the globe wherever animals, mosquitos and people overlap. Blood-feeding mosquitos act as the vector that shuffles viral material between animals and people.

Long’s attention turned from the Sunshine State to Asia in 2012 when Erum Khan, a Pakistani microbiologist with a penchant for arboviruses, recognized her country’s lack of diagnostic tests to confirm an increasing number of fever-based epidemics in the southern Sindh region.

“We had a panic situation in 2013-2014 when a febrile illness surfaced during the religious festival of Eid-ul Azha, and there was confusion about whether it was Congo-Crimean hemorrhagic fever versus other arboviruses,” said Khan, a professor of pathology and laboratory medicine at Aga Khan University in Karachi. “This absolute need to establish the diagnostic capacity resulted in my contacting the Emerging Pathogens Institute for possible collaboration.”

When Pakistani patients exhibit fever combined with neurological symptoms, often a mosquito-borne illness is suspected and, historically, it was often assumed that Japanese encephalitis virus was the culprit. But prior to Long and Khan’s collaboration, many patients were not tested to confirm the cause of their troubles. “Some of our clinicians in rural and urban areas did not have an awareness of when to suspect an arbovirus infection,” Khan said.

With funding from the U.S. Defense Threat Reduction Agency, the duo began to study the spectrum of arboviruses circulating in Pakistan. DTRA is always interested to know what diseases U.S. troops may be exposed to abroad, but part of its mission is also to foster scientific engagement on cooperative biological issues between the U.S. and other countries.

Dr. Erum Khan (second from left, top row) and her team visited U.F. in June 2014 to train in mosquito collection techniques. Photo courtesy of Erum Khan.

Dr. Erum Khan (second from left, top row) and her team visited U.F. in June 2014 to train in mosquito collection techniques. Photo courtesy of Erum Khan

Long was not discouraged by her lack of expertise in making human diagnoses; after all, she was stalking viruses, no matter the species they happened to infect. Plus, she had plenty of experience studying large mammalian hosts—and humans are mammals too.

Because Pakistan lacked a holistic infrastructure for tracking arboviruses—in humans, as well as in mosquito vectors and their animal reservoirs— Long and her co-investigators had the unique opportunity of starting from scratch. “Our single goal was to enroll patients with febrile disease,” Long said. “If a person walked into a hospital in Karachi with a fever, we enrolled them and began testing. Using a broad approach we were able to document more Chikungunya patients with neurological symptoms also. If we only tested the patients with joint pain, neurological symptoms would have been under appreciated, as I suspect happens in many outbreaks of this disease.” 

Long’s study represents the first systematic province-wide effort in Pakistan to measure the prevalence of flaviviruses—a family of disease-causing viruses which includes Zika, dengue, yellow-fever, West Nile and Japanese encephalitis.

The results were eye opening.

Capturing a West Nile virus outbreak

Birds are natural hosts to West Nile virus where it perpetuates a bird-mosquito-bird transmission cycle. But sometimes, several species of mosquito in the genus Culex feed on both birds and people, which allows the virus to jump the species boundary. In people, West Nile virus more often than not causes no symptoms; but in about 20 percent of cases it can create fever, headache, fatigue, nausea, vomiting, body aches, skin rash and swollen lymph nodes. And in less than 1 percent of cases worldwide, severe neurological disease can occur including muscle weakness, tremor or paralysis; a stiff neck, convulsions, altered mental states and even coma or death.

In 2015, Long and Khan launched their study by enrolling patients who came to area hospitals in Karachi, Hyderbad, Mirpurkhas, Larakana, and Sukkur with any combination of unexplained or hemorrhagic fever, or sudden-onset paralysis or muscle weakness. First, they eliminated patients who tested positive for malaria, Crimean-Congo hemorrhagic fever, flu and septicemia and then they screened whoever remained for arboviruses. Right away, samples turned up positive for West Nile virus. 

Maureen Long and graduate assistant using microscope

Post-doctoral assistant Erika Schwarz (left), and Maureen Long (right) examine virus growth in closed tissue plates. The viruses were inactivated and stained with dyes in order to quantify virus in a patient sample. (UF Health Creative Services)

“We went into this expecting to see mostly Japanese encephalitis virus, which is what has been historically present in the Middle East according to all the major sources,” Long said. “But we started getting hits for West Nile immediately. This virus has amazed me in its worldwide expansion and continued outbreaks.”

Between May 2015 and December 2016, the project enrolled 997 patients from all sites and 105 patients tested positive for West Nile virus. From July-December 2015 the study captured a small outbreak of West Nile virus that may have otherwise been attributed to Japanese encephalitis virus.

Although West Nile virus has been known to occur in Pakistan since the 1980s, reliable data for its prevalence is lacking. But for the first time, Long’s study documented neurologically-invasive West Nile virus in Pakistan.

The strain of WNV that causes neurological symptoms arose in New York in 1999 when the virus first caused an outbreak in the United States. Because of the broad parameters for enrolling patients in their study, Long thinks they were able to capture more cases of neurologically-involved West Nile virus than would have otherwise been confirmed. When compared to patients who tested positive for Japanese encephalitis virus, patients who had acquired West Nile virus were more likely to have seizures, encephalitis, hyper excitability, hallucinations, catatonia and stupor.

“The most common neurologic disease in this area from a mosquito would be Japanese encephalitis,” Long said. “I just wonder if it’s being supplanted in this area by West Nile virus.”

Maureen Long and student at hooded vent in BSL

Maureen Long and post-doctoral assistant Erika Schwarz work under a biosafety cabinet that passes air away from people and into a HEPA air filter. This is the only place within a biosafety level III lab where samples can be opened safely in order to perform tests and prepare for virus extraction and molecular identification. (UF Health Creative Services)

The project also trapped more than 8,900 mosquitos throughout Karachi to survey what kinds of mosquitos were present, and how their numbers fluctuated in tangent with air temperature. After a record-breaking heat wave was omitted, mosquito abundance correlated with higher air temperatures; and the human cases of West Nile virus lagged a few weeks behind as would be expected given the incubation period.

Their insect catch was dominated by Culex mosquitos, with just a few Aedes species showing up.  Culex is the main driver of West Nile virus, while certain Aedes species spread Chikungunya. Long thinks the proportional discrepancy in types of mosquitos found was an artifact of the traps themselves and that there were likely far more Aedes mosquitos present than the study found.

The presence of West Nile virus in Pakistan is best understood in the context of bordering countries. Within the past few years, the virus has been detected in Iran, Afghanistan and India where it has infected people, horses, domestic and wild birds, and Culex mosquitoes – and it’s even been identified as the cause of human encephalitis cases.

The only reason it had gone unreported in Pakistan is simply because no one was looking.

Chikungunya: The virus that stole Christmas

About a year after the West Nile outbreak, a second epidemic began sweeping Karachi in November and December 2016. Warm temperatures reaching the mid 70s to low 80s are becoming typical in these months as the region experiences climactic shifts to warmer winters.

“A couple of days before Christmas, Erum emailed that she had to bring her team in, they were seeing really bad febrile arthritis and they suspected Chikungunya,” Long said. Their suspicions proved correct, and the fever-surveillance study happened to capture a second epidemic in real time. Over the study period, 102 of 997 patients tested positive for Chikungunya. Other health entities in the country confirmed more than 4,000 cases in Karachi that winter and it’s thought that more than 30,000 people were sickened.

Chikungunya is more symptomatic in people than is West Nile virus, and in addition to fever it can cause severe joint and muscle pain. Similar to West Nile virus, the study found neurological symptoms in up to 49 percent of the Chikungunya-infected patients. This was the first time neurologically-involved Chikungunya was widely documented in an Asian country. It had previously been thought of only as a rare complication. “But as you can see, our finding was that it is not at all rare,” Long said.

Around 21.6 percent of Chikungunya patients in the study experienced encephalitis, with 5 percent experiencing a severe nerve-damaging form known as acute disseminated encephalomyelitis. Although Chikungunya-infected patients were just as likely as sufferers of West Nile virus to experience neurological symptoms and encephalitis, they were 4.5 times more likely to experience abnormally low levels of lymphocytes—specialized white blood cells with important immune functions.

Pakistan is ranked as highly vulnerable to effects of climate change. Long term trends of a warming climate make it perfectly hospitable to pathogen-carrying insects. Infrastructure projects such as superhighways and shipping ports facilitating movement of people and goods to and from Africa and China increase the likelihood of novel pathogens being imported. And the lack of a comprehensive screening process for blood donation creates another mode of transmission to new human hosts.

Long and Khan want to build scientific infrastructure to help Pakistan face a changing future, one that is all but certain to bring waves of mosquito-borne illnesses.

Participants from urban and rural cities of Pakistan's Sindh region trained at Aga Khan University in Karachi between May 8-10th in 2018 to learn about arbovirus field epidemiology, laboratory diagnosis and vector analysis. Photo courtesy of Erum Khan.

Participants from urban and rural cities of Pakistan's Sindh region trained at Aga Khan University in Karachi between May 8-10th in 2018 to learn about arbovirus field epidemiology, laboratory diagnosis and vector analysis. Photo courtesy of Erum Khan.

Scientific exchange: Training virus stalkers

Long and Khan envision permanently implementing the arbovirus surveillance system they devised for their study, not only for research’s sake, but for the health and welfare of the Pakistani people. While a typical research study would gather data, analyze and publish once the funding ran out, Long saw the chance to do something different under DTRA’s rubric of cooperative biological engagement. She envisions a Pakistani entity that can detect and respond to all facets of interactions between an arbovirus, its insect vector, its animal and human hosts, and the climate and habitat in which it persists.

Interactive live web training. Photos courtesy of Erum Khan.

Participants at Aga Khan University setting up a live web training session. (Photo courtesy of Erum Khan)

Although the original project called for on-the-ground training in Pakistan, geopolitical events led to the State Department changing Pakistan’s travel advisory status unfavorably. “We had to be clever, as a result,” Long said. They used two-way video feeds for live web-based conferences and lab-safety training programs. “The participants in Pakistan would be working under a lab hood with agents, and we would be telling them what to do each step of the way," Long said. "They could watch us, and we could watch them. It was highly interactive.”

Khan trained 70 healthcare workers (doctors, lab technicians, pathologists and students plus several fellows and post-doctoral students) in how to screen and enroll patients in the study, how to suspect specific arboviruses, and the lab work to analyze blood samples. “We ran lectures, and Dr. Khan did too, and trained people throughout the Sindh province. Which was kind of cool because it made a network, and that is how science should work,” Long said.

Pakistani health professionals from urban and rural cities receiving interactive web training on arbovirus detection and diagnosis at Aga Khan University. Photos courtesy of Erum Khan.

A training workshop at Aga Khan University in Karachi. (Photo courtesy of Erum Khan)

“On our side, we set out to train Pakistani scientists and laboratory personnel, and then have this technology transferred to Pakistan with the goal of the Karachi laboratory and hospital laboratory becoming proficient within three to five years,” Long said. “Dr. Khan and her team are amazing. This season, arbovirus testing will likely be routine in the hospital microbiology laboratory at Aga Khan in Karachi." Long’s lab in the U.S. will continue to act as a reference lab to validate the results and the skill set of her Pakistani collaborators. And while they crystalize their surveillance system, Long is turning her attention to the mechanisms of how West Nile virus causes disease.

“I’m not a good traveler,” Long confessed; she still has not visited Pakistan, although it is a future goal. But that has not dampened her drive to work globally. “This project has become a passion, it’s been one of the best experiences of my career. Through today’s technology we were able to interact constantly rather than spending short periods of time in-country. I think in the end our professional relationship has been much closer.”

Long plans to continue remotely-based research engagement in African and middle Eastern countries where scientific infrastructure has been damaged by war.

Maureen Long and student

Maureen Long, right, deeply enjoys teaching and mentoring BSLIII lab safety techniques to students and colleagues such as Schwarz, on the left. (UF Health Creative Services)


Creator credits

Written by DeLene Beeland. Special thanks to Dr. Maureen Long for her participation and time, and to UF Health Creative Services photographer Jesse Jones for creating photos of Dr. Long in her lab. And a very special thanks to Dr. Erum Khan for sharing photos and ideas from Pakistan.

 

Read more

This article is based on the following published literature:

Evidence of Chikungunya Virus Disease in Pakistan Since 2015 With Patients Demonstrating Involvement of the Central Nervous System. Frontiers in Public Health. 2018 Jul 10;6:186. doi: 10.3389/fpubh.2018.00186.

Human West Nile Virus Disease Outbreak in Pakistan, 2015-2016. Frontiers in Public Health. 2018 Feb 27;6:20. doi: 10.3389/fpubh.2018.00020.

Flaviviruses as a Cause of Undifferentiated Fever in Sindh Province, Pakistan: A Preliminary Report. Frontiers in Public Health. 2016 Feb 16;4:8. doi: 10.3389/fpubh.2016.00008