June 2008
Feature Story

William Check, PhD

English geologist Sir Charles Lyell in 1830 postulated the doctrine of uniformitarianism, which held that slow-moving forces acting over long periods shaped the Earth. Mountain ranges rose and were worn down over tens to hundreds of millions of years. Compared with geological epochs, epidemiologically important changes in the landscape of viral pathogens can be observed in mere eyeblinks. In the past 40 years we have seen the emergence of five devastating or potentially devastating viruses or groups of viruses: the highly lethal filoviruses Ebola and Marburg, arenaviruses such as Lassa in Africa and Machupo in South America, human immunodeficiency virus, the coronavirus that causes severe acute respiratory syndrome, and an H5 variant of influenza virus that many experts believe could evolve to cause a pandemic worse than the 1918 pandemic.

On an even shorter time scale, we are confronted with viral pathogens that are less apocalyptic. Since the start of this century, several such threats have arisen that are all “new” in some important sense and that cause human illness of varying frequency and severity:

Human bocavirus (HBoV), a previously unknown virus, was first reported in 2005 as a result of a systematic search for undiscovered viral respiratory pathogens.

Human metapneumovirus (hMPV), also a previously unknown virus, was first isolated from patients in 2001, also from a systematic search.

Nipah and Hendra, two newly emergent viruses first encountered in Malaysia and Australia, respectively, in the mid- and late 1990s, cause a high case-fatality rate and together define a new genus of paramyxoviruses.

Chikungunya virus, which has been known since 1953 to be endemic to Africa and the Indian Ocean, was found in 2006 and 2007 in scores of travelers returning from these areas to the United States and to several Western European countries.

Adenovirus 14, a rare serotype of adenovirus species B2 that was first diagnosed in the United States in 2003, has already caused outbreaks of sometimes severe respiratory disease in several states.

Coxsackievirus B1 was the predominant enterovirus serotype in the United States in 2007 (MMWR. 2008; 57:553–556).

Norovirus has in the past several years been increasingly appreciated as the chief cause of outbreaks of gastroenteritis, not only on cruise ships but also in the community and in long-term care facilities.

Important clinical and diagnostic data on all of these emerging, evolving, or emigrating viruses were presented in April at this year’s Clinical Virology Symposium and Pan American Society for Clinical Virology meeting. Interviews with several scientists involved in the work presented at the meeting, and with others who study emerging viruses, sketch a picture of the geography and topography of contemporary clinical virology.

One of the most fundamental questions is what these ongoing upheavals in the viral landscape signify. The answer, it turns out, depends on the virus. With regard to discovery of truly new, previously unknown viruses, Tobias Allander, MD, PhD, who was responsible for the discovery of human bocavirus, believes we are only at the beginning. “There haven’t previously been good techniques to search for viruses in a comprehensive way,” explains Dr. Allander, associate professor and clinical virologist in the Department of Clinical Microbiology, Karolinska University Hospital, Stockholm. “We could realize that because new viruses are discovered, but not reproducibly, there is a pool of unknown viruses to pick from. When you work in clinical virology, you realize that for a high proportion of patients you never find any agent.”

James E. Crowe Jr., MD, who has done work on the clinical importance of human metapneumo­virus, agrees that the discovery of genuinely new viruses represents for the most part an improvement in molecular diagnostic techniques. “It probably does not represent a significant change in the epidemiology of diseases,” says Dr. Crowe, who is Ingram professor of cancer research in the Division of Infectious Diseases, Department of Pediatrics, Vanderbilt University Medical Center. The unearthing of new pathogenic viruses will “explode” in the next few years, Dr. Crowe predicts. “I would anticipate hundreds of new viruses being discovered,” he says, which will greatly reduce the proportion of illnesses with no identifiable etiology.

Addressing a different category of new viruses, those like Nipah and Hendra that became known as a result of lethal outbreaks, William J. Bellini, PhD, says, “The emergence of these viruses is exactly that. They probably have been around forever. It is only when humans come face to face with vectors or carriers of these viruses in a sort of perfect storm that you see them crop up.” For the emergence of Nipah in Malaysia, for instance, “It was extension of pig farms that apparently forced pigs into a situation where they were in juxtaposition to roosting bats and made a connection that never had been made before,” says Dr. Bellini, chief of the Measles, Mumps, Rubella, and Herpesvirus Laboratory Branch, Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention.

Some scientists assert that “our raping of the environment may in fact be getting us into situations that are better left alone,” he says. “There is something to be said for [the dangers of] people extending residential or farming areas where they had never been before.”

Chikungunya virus is perhaps a bit different than SARS or some other viruses, says Robert S. Lanciotti, PhD, chief of the Diagnostic and Reference Laboratory in the Arbovirus Diseases Branch, CDC, Fort Collins, Colo. “It is actually a very old virus that was discovered in Africa. When people were looking for yellow fever, they found lots of interesting viruses, including chikungunya and West Nile.” Dr. Lanciotti says the biggest factor in the emergence of chikungunya is global travel.

“Viruses used to have a very narrow geographic range, primarily due to limitations of travel,” he says. Even the mosquito vector, Aedes albopictus, used to be found in a fairly limited geographic area. However, because of transportation of tires and raw materials, this mosquito is now found in the western hemisphere. In addition, chikungunya appears to have adapted to grow more efficiently in Ae. albopictus.

Viruses are both exciting to study and a potential threat for emerging agents because they are genetically simple and have a lot of room to change, says David Schnurr, PhD, research scientist supervisor II in the California Department of Public Health’s Viral and Rickettsial Disease Laboratory, Richmond. “New strains and types can appear very quickly,” Dr. Schnurr says, “and there is a great reservoir in the wild.” And zoonotic diseases, such as SARS and HIV, have the potential to cross into humans, he notes.

Steven Specter, PhD, associate dean for student affairs and professor of molecular medicine at the University of South Florida College of Medicine, Tampa, and chair of the Clinical Virology Symposium, says the emergence of new agents or “our newer understanding of the pathogenic roles of already known agents—which people often refer to as reemergence—is of great interest literally to all microbiologists and infectious disease physicians because of its clinical implications.” Dr. Specter agrees that this is driven by molecular techniques that are more sensitive and by our incursion into areas we previously did not enter—rain forests and other harsh environments.

“Another part of this puzzle is that we are doing more now in trying to deal with health problems in resource-limited areas like Asia and Africa,” he says. “These viruses have probably been around for a long time but no one recognized them as causes of disease because no one tried to detect them.”

Dr. Specter cites two examples of new viruses that are even more recent: a lethal arenavirus in transplant patients discovered through unbiased high-throughput sequencing (Palacios G, et al. N Engl J Med. 2008; 358: 991– 998) and the Torque Teno virus, which does not have a clear disease association but may cause hepatic dysfunction or damage in transfusion-dependent thalassemia patients, or both (Hu YW, et al. J Med Virol. 2008; 80: 365– 371).

As these emerging viruses are seen, Dr. Specter says, “they tell us that this problem is going to continue. Being aware of ‘zebras’ is an important part of the job of clinical laboratories.” As Dr. Bellini puts it, “Always be vigilant.” Laboratorians must constantly monitor the spectrum of circulating viral pathogens. “You have new and emerging viruses to worry about and old and just about eradicated viruses that you still can’t forget about,” Dr. Bellini says. (See “The return of measles, mumps, and rubella,” page 94.) “So clinicians and clinical lab people have a lot to think about. It seems like you are juggling a thousand balls all the time.”

For a closer look at the emergence and clinical significance of each of these new viral pathogens, let’s start with one of the truly new ones, human bocavirus, or HBoV. “When we look for new agents, we find very old agents that have been around for a long time,” Dr. Allander says. “I think that human bocavirus is a very old virus—perhaps thousands of years—that is very well adapted to humans.” HBoV was isolated from samples obtained from patients with respiratory tract disease in Stockholm (Allander T, et al. Proc Natl Acad Sci USA. 2005; 102: 12891– 12896). Dr. Allander’s group obtains virus particles from pooled patient samples and this is followed by cloning, large-scale sequencing, and then bioinformatic analysis. “Selectivity for viral sequences is very much in the computer software,” Dr. Allander says. “We let the computer decide what is a virus.” This approach has become more feasible with the availability of more sequences in databanks and the ability to sequence genomes at modest cost that has derived from the Human Genome Project. Last year they reported the discovery of a new polyomavirus using this method (Allander T, et al. J Virol. 2007; 81: 4130– 4136).

Although HBoV was isolated from children with respiratory disease, “that doesn’t automatically mean the virus was responsible for that disease,” Dr. Allander cautions. In many people well-adapted viruses such as HBoV are carried without symptoms. HBoV clearly seems to be a major respiratory pathogen, but establishing its pathogenicity is a long process that Dr. Allander says has not yet been formally completed. With a virus like HBoV that cannot yet be cultured, Koch’s postulates—cultivating the virus and inoculating it into animals—can’t be fulfilled. Indirect evidence has to be provided from diagnostic studies. As one step in that process, at the Clinical Virology Symposium Dr. Allander presented data on two children in whom HBoV was strongly linked to serious disease and no other isolate was found. One patient was a four-year-old girl with a history of infection-related wheezing who suffered a life-threatening episode of acute wheezing, a symptom consistent with published epidemiologic data (Allander T, et al. Clin Infect Dis. 2007; 44: 904– 910). A U.S. study found HBoV in about five percent of children’s respiratory specimens that tested negative for common viral respiratory pathogens (Kesebir D, et al. J Infect Dis. 2006; 194: 1276– 1282). None of 96 asymptomatic children were positive for HBoV. At least half of HBoV-positive children in this study had wheezing.

Proving causality has been complicated by post-infectious shedding of HBoV, Dr. Allander says. “[M]any patients have presented with low viral DNA loads, suggesting HBoV persistence and rendering polymerase chain reaction-based diagnosis without quantitative analysis problematic,” he and colleagues wrote (Kantola K, et al. Clin Infect Dis. 2008; 46: 540– 546). They found that “Serological diagnoses correlate with high virus loads in the nasopharynx and with viremia.” A stringent algorithm for HBoV diagnosis includes PCR on blood samples and IgM serology on positive samples to establish recent infection. Dr. Allander’s clinical laboratory began in October 2007 to include testing for HBoV in its complete respiratory panel.

The other truly new virus, the paramyxovirus human metapneumo­virus, or hMPV, was also discovered with an advanced molecular technique, fingerprinting by RNA arbitrarily primed PCR (RAP-PCR) from nasopharyngeal aspirates of Dutch children with respiratory symptoms (van den Hoogen BG, et al. Nat Med. 2001; 7: 719– 724). All samples showed cytopathic effect on cell monolayers but tested negative by all of the laboratory’s routine techniques. In a subsequent study of the virus’ clinical role, the Dutch group detected hMPV RNA in seven percent of samples from patients with respiratory tract illnesses, primarily in very young children and in immunocompromised individuals (van den Hoogen BG, et al. J Infect Dis. 2003; 188: 1571– 1577). They concluded: “hMPV is an important pathogen associated with [respiratory infections].”

The clinical role of hMPV was not at first universally accepted because its seasonality and symptoms overlap with those of respiratory syncytial virus, or RSV. The two viruses are often found together in patient samples. Now its clinical relevance is recognized. “Throughout the world,” Dr. Crowe says, “people looked at samples coming through diagnostic laboratories, either bronchoalveolar lavage or nasal washes. Human metapneumovirus was present in five to 10 percent of samples in winter and not present in summer.” In a study that Dr. Crowe led, hMPV was often found as the only agent identified (Williams JV, et al. N Engl J Med. 2004; 350: 443– 450). “That’s really the strongest evidence,” he says. Other researchers have verified this finding. Dr. Crowe and his colleagues concluded: “Human meta­pneumovirus infection is a leading cause of respiratory tract infection in the first years of life. . . .” Twelve percent of lower respiratory infections in infants and children were attributed to hMPV. Disease severity mirrors that of RSV infection (Mullins JA, et al. Emerg Infect Dis. 2004; 10: 700– 705). Coincident positive molecular tests for hMPV and RSV are not more frequent than expected on the basis of probability, Dr. Crowe says. Also, the pathogenicity of hMPV has been demonstrated in small animals and non-human primates.

“The respiratory virus research community is very aware of this pathogen,” Dr. Crowe notes. “It appears to be the second most important cause of wheezing and pneumonia in young children. However, the clinical community is not particularly aware of it given its impact on children’s health.” The main reason for this, in Dr. Crowe’s view, is that there is no licensed diagnostic test. “As physicians, we can’t pay attention to things for which we don’t have a good test,” he says. He predicts there will soon be diagnostic tests for hMPV. When that happens, the same patients who are candidates for RSV testing will be candidates for hMPV testing. In the meantime, testing for hMPV can be ordered from commercial laboratories as part of a panel.

“Sometimes people say, ‘If I had a test for hMPV, what would I do?” Dr. Crowe says. He points to more appropriate isolation procedures. All children with wheezing are typically housed in one area of a hospital, sometimes on wards with multiple patients in a room. “Now we know we might be grouping children with hMPV and RSV, so we could be putting patients at risk of nosocomial infections,” he says. A test for hMPV could make more effective isolation possible.

Young children aren’t the only ones affected by hMPV. Dr. Schnurr and colleagues recently reported a summer outbreak of hMPV in a long-term care facility (Louie JK, et al. J Infect Dis. 2007; 196:705–708).

When Nipah virus first emerged in Malaysia and Singapore in 1999, Dr. Bellini says, it had an intermediate host in pigs. “We thought that perhaps the reason this cropped up in pigs was that pig farms, which form a large portion of the Malaysian economy, at that time had started to encroach on areas that were previously not utilized by Malaysians who live there,” Dr. Bellini says. Pig farms arose in jungle areas where large fruit bats, also known as flying foxes (Pteropus sp), roosted in fruit orchards. More than 100 cases were identified; they were either pig farmers handling pig urine and feces or abattoir workers who slaughtered pigs.

Later outbreaks that occurred in Bangladesh in 2004 and almost yearly since then are smaller, and no intermediate veterinary host has been identified. Those who are infected suffer rapidly progressive severe illness affecting the central nervous and respiratory systems (Bellini WJ, et al. J Neurovirol. 2005; 11: 481– 487; Hossain MJ, et al. Clin Infect Dis. 2008; 46: 977– 984). In the Bangladeshi outbreaks, Dr. Bellini says, “we think there was direct bat-to-human transmission.” Fruit bats were observed to be in contact with an extract harvested from date palms as a consumable beverage, though “direct isolation of Nipah virus from that source has not been verified and it is still basically conjecture how humans came into contact with the virus,” Dr. Bellini says. “Human-to-human spread has been implicated in health care settings such as the outbreaks in the Faridpur District of Bangladesh or Siliguri [West Bengal]” (Gurley ES, et al. Infect Control Hosp Epidemiol. 2007; 28: 740– 742; Chadha MS, et al. Emerg Infect Dis. 2006; 12: 235– 240). In those instances, close contact with patients who were not placed in isolation and where hospital infection control was suboptimal led to spread of the disease. “The real question,” Dr. Bellini says, “is why human-to-human spread was not observed in the Malays­ian/Singa­pore outbreak.” One speculation is that excellent hospital infection control was responsible. No substantive genetic changes in the Nipah viruses involved in the different outbreaks have been identified.

Dr. Bellini notes that Nipah occurs only in the Pteropus flyway. “But that flyway is pretty vast,” he says, “from Madagascar and the east coast of Africa to Australia into Asia, the foothills of the Himalayas and regions of Indonesia and the eastern Pacific.”

Dr. Lanciotti’s laboratory at the CDC in Fort Collins was involved directly in diagnosing chikungunya infection in travelers returning from India or islands in the Indian Ocean during the 2005–2006 epidemics there. When returning travelers presented with illness compatible with chikungunya infection, samples ended up at his laboratory, since other laboratories were not equipped to test for this virus. “That is what we do here,” Dr. Lanciotti says. “We work with what some people call exotic viruses—viruses that are rare in the western hemisphere.” About 38 cases were eventually detected (Lanciotti RS, et al. Emerg Infect Dis. 2007; 13:764– 767; MMWR. 2007; 56: 276– 277).

Chikungunya has a particular clinical presentation—acute onset of arthritis. “There are not many infectious causes of that,” Dr. Lanciotti says. His laboratory performs serology for specific IgM (which persists for three to four weeks) to identify recent infection. “We also do PCR to see whether there is circulating virus,” he says. Quantitative PCR testing raised concern. “We have a long history here of working with mosquitoes and know the concentration of virus required to infect mosquitoes,” Dr. Lanciotti says. “Many people coming back to the U.S. had live virus circulating in quantities high enough to infect the mosquito population for introduction of chikungunya into the U.S.” Resident U.S. mosquitoes are susceptible to infection with chikungunya (Reiskind MH, et al. Am J Trop Med Hyg. 2008; 78:422–425).

However, although local transmission of chikungunya did occur in Italy (Rezza G, et al. Lancet. 2007; 370: 1840– 1846), it did not happen in the United States. Nor were infected mosquitoes detected. “That makes sense,” Dr. Lanciotti says. Most travelers returned to the United States in late fall and winter, times not conducive to establishing the virus in mosquitoes. Or they came back to places where there is no Ae. albopictus—the primary vector of chikungunya in the United States. (Its range is mostly in the southeast U.S.)

“The bottom line is that we dodged a bullet this time around,” Dr. Lanciotti says. “But as we have seen with other viruses, like West Nile, it just takes the correct conditions. We know the other side of the story.”

A small number of commercial laboratories have now established chikungunya testing or are in the process. “They have contacted us for controls,” Dr. Lanciotti says. “At this point I don’t think it would be efficient for a lot of labs to establish testing for chikungunya. If local transmission occurred, our first action would be to equip and train state public health labs, as we did with West Nile. Then commercial labs would set up the test. For local labs, the best plan of action would be to send specimens to us.”

In contrast to exotic viruses, adeno-virus is a well-known pathogen. “It’s a DNA virus so it’s pretty stable,” Dr. Schnurr says. Adenovirus serotype 14 (Ad14) was first seen among military recruits in the Netherlands in 1955; outbreaks occurred in Europe until the early 1960s, then it disappeared. Ad14 caused an outbreak of respiratory disease among children in Taiwan in 2001. “It first showed up in the United States in 2003,” Dr. Schnurr says. “Our lab detected that case, but seeing one case didn’t tell us how important it would become. It has now emerged as a cause of ongoing outbreaks of respiratory disease among military recruits throughout the U.S. and in some civilian populations.”

A single case was reported in New York, and clusters have been recognized in Oregon, Texas, and Washington (MMWR. 2007; 56: 1181– 1184). Unlike typical adenovirus-caused disease, some people have become severely ill—38 percent of patients in the MMWR report were hospitalized. “The cases described in this report are unusual because they suggest the emergence of a new and virulent Ad14 variant that has spread within the United States,” the MMWR report said. Based on an investigation of three patients in California, Dr. Schnurr and colleagues concluded: “Our identification of severe respiratory illness due to a previously rarely reported adenovirus serotype may signify the emergence in the United States of a new genomic variant that has the potential to spread globally and cause epidemics” (Louie JK, et al. Clin Infect Dis. 2008; 46: 421–425).

As to why Ad14 has emerged, Dr. Schnurr says there is evidence for changes in its genome that allow it to grow much more readily in human respiratory tissue, and that may have increased its virulence.

In a report at the Clinical Virology Symposium, Steven Oberste, PhD, chief of the Picornavirus Laboratory in the National Center for Immunization and Respiratory Diseases, said an unusually high number of cases of severe neo­natal illness were reported to the CDC in 2007—514 cases from 36 states. Of the 514 cases reported, 444 were of known serotype. Coxsackievirus B1 (CVB1) was responsible for one-fourth, or 113, of these 444 cases. (These 113 CVB1 cases were reported from 19 states.) Five of the 113 CVB1 cases were fatal. Fatal cases were part of local outbreaks of CVB1-associated severe neonatal illness. In 2007, for the first time, CVB1 became the most prevalent serotype in the United States. Genetic analysis showed “possible recent emergence and spread of a new gen­etic lineage [of CVB1],” Dr. Oberste and colleagues suggested. “Un­fortunately,” he says, “there is no specific inter­vention for this virus,” only aware­ness and hygiene.

Says Dr. Schnurr, “We first learned about this through an ongoing outbreak of enterovirus infection in newborns in Alaska.” His laboratory helped identify, by PCR, one of the cases of myocarditis as due to an enterovirus. In the ongoing CVB1 outbreak around the United States, severe meningitis and myocarditis are being seen. “Exactly why B1 is now emerging as a major epidemic virus in the last year, I don’t think anyone has a handle on that,” Dr. Schnurr says.

Several reports at the Clinical Virology Symposium described the development of molecular assays for norovirus, which Dr. Schnurr says has emerged in the past few years as the primary cause of outbreaks of gastroenteritis. “Going back 10 years we didn’t have the tools to diagnose norovirus infections very effectively,” he notes. Norovirus still has not been grown in culture, which makes it difficult to study. “Now molecular tools—sequencing and PCR—have allowed us to learn a great deal about the outbreaks that norovirus causes,” Dr. Schnurr says. “It is exciting and pretty much a new field.” It has been discovered that norovirus is responsible for 70 to 80 percent of classical gastroenteritis outbreaks; in long-term care facilities that figure rises to 90 percent.

Norovirus gastroenteritis is almost always of fairly short duration—24 to 48 hours—and it doesn’t have sequelae. However, viral shedding can continue for up to two weeks. “The main thing is to limit spread among food handlers and to keep children away from school,” Dr. Schnurr says. In long-term care facilities isolation is needed.

As viral pathogens proliferate with no apparent limit, laboratory professionals face a diagnostic challenge, Dr. Lanciotti says. “Our challenge is to move in the direction of very broad assays that are able to pick up a lot of different viruses,” he says. “To me the most logical way is something like microarrays.” Dr. Lanciotti cites as one example the pan-­viral DNA microarray Virochip, which bears the most conserved sequences of all known viruses. It was developed at the University of California, San Francisco.

“That is the direction that I think reference laboratories are going to have to go in to be prepared to test specimens for multiple pathogens,” he says. “Rather than dividing a patient sample into 100 different tests, we can use one specimen and screen for hundreds of viruses. That is the need in diagnostic virology right now, and you need virologists to do that.”

However, this task will not be easy. Ribosomal RNA can be used as a universal probe for bacteria. For viruses there is no current obvious candidate. “That is the part of this that needs the most work,” Dr. Lanciotti says.

Containment is another important element in addressing emerging viruses. Dr. Specter cites SARS as a potentially tremendous epidemic that got cut off because of cooperation between the World Health Organization and the CDC and communication to physicians and laboratories. “SARS emerged, was important for a year or two, and has now totally gone away,” he says. “It provides a very important lesson in how to interrupt potentially disastrous epidemics.” Dr. Specter cites two key factors: first, disseminating information to infectious disease physicians to make them more aware so they could take precautions and, second, spread of information through the Laboratory Response Network, which made it easier to identify cases of SARS infection because they knew what to look for. “Both of those factors were critical,” Dr. Specter says.

He adds one more element that was key to recognizing a dangerous epidemic problem—when health care professionals became ill. “That showed human-to-human transmission,” he says. “For avian flu, we will find out when we get a strain that converts from zoonotic to human-to-human transmission when it arises in health care professionals. That is something that lab professionals need to understand to be able to take precautions early.”

Related Links

• The return of measles, mumps, and rubella.