Adenovirus 14 - CDC: New Respiratory Bug Has Killed 10

[hawkeye]

CDC: New Respiratory Bug Has Killed 10
ATLANTA (AP) - A mutated version of a common cold virus has caused 10 deaths in the last 18 months, U.S. health officials said Thursday.

Adenoviruses usually cause respiratory infections that aren't considered lethal. But a new variant has caused at least 140 illnesses in New York, Oregon, Washington and Texas, according to a report issued Thursday by the U.S. Centers for Disease Control and Prevention.

The illness made headlines in Texas earlier this year, when a so- called boot camp flu sickened hundreds at Lackland Air Force Base in San Antonio. The most serious cases were blamed on the emerging virus and one 19-year-old trainee died.

"What really got people's attention is these are healthy young adults landing in the hospital and, in some cases, the ICU," said Dr. John Su, an infectious diseases investigator with the CDC.

There are more than 50 distinct types of adenoviruses tied to human illnesses. They are one cause of the common cold, and also trigger pneumonia and bronchitis. Severe illnesses are more likely in people with weaker immune systems.

Some adenoviruses have also been blamed for gastroenteritis, conjunctivitis and cystitis.

There are no good antiviral medications for adenoviruses. Patients usually are treated with aspirin, liquids and bed rest.

In the CDC report, the earliest case of the mutated virus was found in an infant girl in New York City, who died last year. The child seemed healthy right after birth, but then became dehydrated and lost appetite. She died 12 days after she was born.

Tests found that she been infected with a form of adenovirus, called Ad14, but with some little differences, Su said.

It's not clear how the changes made it more lethal, said Linda Gooding, an Emory University researcher who specializes in adenoviruses.

Earlier this year, hundreds of trainees at Lackland became ill with respiratory infections. Tests showed a variety of adenoviruses in the trainees, but at least 106?and probably more?had the mutated form of Ad14, including five who ended up in an intensive care unit

In April, Oregon health officials learned of a cluster of cases at a Portland-area hospital. They ultimately counted 31 cases, including seven who died with severe pneumonia. The next month, Washington state officials reported four hospitalized patients had the same mutated virus. One, who also had AIDS, died.

The Ad14 form of adenovirus was first identified in 1955. In 1969, it was blamed for a rash of illnesses in military recruits stationed in Europe, but it's been detected rarely since then. But it seems to growing more common. The strain accounted for 6 percent of adenovirus samples collected in 22 medical facilities in 2006, while none was seen the previous two years, according to a study published this month in the medical journal Clinical Infectious Diseases.

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5645a1.htm

Acute Respiratory Disease Associated with Adenovirus Serotype 14 --- Four States, 2006--2007



Adenovirus serotype 14 (Ad14) is a rarely reported but emerging serotype of adenovirus that can cause severe and sometimes fatal respiratory illness in patients of all ages, including healthy young adults. In May 2006, an infant in New York aged 12 days died from respiratory illness caused by Ad14. During March--June 2007, a total of 140 additional cases of confirmed Ad14 respiratory illness were identified in clusters of patients in Oregon, Washington, and Texas. Fifty-three (38%) of these patients were hospitalized, including 24 (17%) who were admitted to intensive care units (ICUs); nine (5%) patients died. Ad14 isolates from all four states were identical by sequence data from the full hexon and fiber genes. However, the isolates were distinct from the Ad14 reference strain from 1955, suggesting the emergence and spread of a new Ad14 variant in the United States. No epidemiologic evidence of direct transmission linking the New York case or any of the clusters was identified. This report summarizes the investigation of these Ad14 cases by state and city health authorities, the U.S. Air Force, and CDC. State and local public health departments should be alert to the possibility of outbreaks caused by Ad14. New York


In May 2006, a fatal case of Ad14 illness occurred in New York City in an infant girl aged 12 days. The infant was born after a full-term pregnancy and uncomplicated delivery. She was found dead in bed, where she had been sleeping. The infant had been examined 3 days after birth and noted to have lost weight but was otherwise healthy. The next week she had decreased tears with crying, suggesting early dehydration. Physical activity and feeding progressively decreased during the week before her death.
Postmortem tracheal and gastric swabs from the infant were sent to the Wadsworth Center laboratory of the New York State Department of Health, where adenovirus was detected by polymerase chain reaction (PCR). Adenovirus also was isolated by culture, confirmed by immunofluorescence assay (IFA), and typed as Ad14 by antibody neutralization assay. Analysis at CDC identified the same unique genetic sequences in this isolate as were later identified in the Ad14 isolates from the three 2007 clusters.

Autopsy and histologic findings at the Office of the Chief Medical Examiner in New York City included presence in the lung of chronic inflammatory cells with intranuclear inclusions, consistent with adenoviral bronchiolitis and acute respiratory distress syndrome. Investigation by the New York City Department of Health and Mental Hygiene has not identified any other local cases of Ad14 illness. Oregon


In early April 2007, a clinician alerted the Oregon Public Health Division (OPHD) regarding multiple patients at a single hospital who had been admitted with a diagnosis of severe pneumonia during March 3--April 6. A total of 17 specimens were obtained from patients; 15 (88%) yielded isolates that were identified by CDC as Ad14. Through retrospective examination of laboratory reports from the three clinical laboratories in the state that have virology capacity and the Oregon State Public Health Laboratory (OSPHL), OPHD identified 68 persons who tested positive (by culture, PCR, or IFA) for adenovirus during November 1, 2006--April 30, 2007. Isolates from 50 (74%) of these patients were available for further adenovirus typing at either CDC or OSPHL. Of the 50 patient isolates, 31 (62%) were identified as Ad14, and 15 (30%) were identified as another adenovirus type (Figure); four (8%) did not test positive for adenovirus.
Among 30 Ad14 patients (i.e., all but one) whose medical charts were reviewed, 22 (73%) were male; median age was 53.4 years (range: 2 weeks--82 years). Five cases (17%) occurred in patients aged <5 years, and the remaining 20 (83%) occurred in patients aged >18 years. Twenty-two patients (73%) required hospitalization, sixteen (53%) required intensive care, and seven (23%) died, all from severe pneumonia. Median age of the patients who died was 63.6 years; five (71%) were male. One death occurred in an infant aged 1 month. Of the 30 Ad14 cases with patient residence information available, 28 (93%) occurred in residents of seven Oregon counties, and two cases occurred in residents of two Washington counties. No link was identified in hospitals or the community to explain transmission of Ad14 from one patient to another.

In comparison with the Ad14 patients, among the 12 adenovirus non-type 14 patients (i.e., all but three) whose medical charts were reviewed, nine (75%) were male. Median age was 1.1 years, and 11 (92%) patients were aged <5 years. Two (17%) adenovirus non-type14 patients required hospitalization; no ICU admissions or deaths were reported in this group. Washington


On May 16, 2007, the Tacoma-Pierce County Health Department notified the Washington State Department of Health (WADOH) of four residents housed in one unit of a residential-care facility who had been hospitalized recently for pneumonia of unknown etiology. The patients were aged 40--62 years; three of the four were female. One patient had acquired immunodeficiency syndrome (AIDS); the three others had chronic obstructive pulmonary disease. All four were smokers.

The patients had initial symptoms of cough, fever, or shortness of breath during April 22--May 8, 2007. Three patients required intensive care and mechanical ventilation for severe pneumonia. After 8 days of hospitalization, the patient with AIDS died; the other patients recovered. Respiratory specimens from all four patients tested positive for adenovirus by PCR at the WADOH laboratory; isolates were available from three patients, and all three isolates were identified as Ad14 by CDC. Ad14 had last been identified in an isolate from a patient from Washington in May 2006, marking the first identification of Ad14 in the state since 2004. Active surveillance among facility residents and staff did not identify any other cases of Ad14 illness. Texas


Since February 2007, an outbreak of cases of febrile respiratory infection* associated with adenovirus infection has been reported among basic military trainees at Lackland Air Force Base (LAFB). During an initial investigation, conducted from February 3 to June 23, out of 423 respiratory specimens collected and tested, 268 (63%) tested positive for adenovirus; 118 (44%) of the 268 were serotyped, and 106 (90%) of those serotyped were Ad14. Before this outbreak, the only identification of an Ad14 isolate at LAFB occurred in May 2006 (1).
During February 3--June 23, 2007, a total of 27 patients were hospitalized with pneumonia (median hospitalization: 3 days), including five who required admission to the ICU. One ICU patient required extracorporeal membrane oxygenation for approximately 3 weeks and ultimately died. All 16 hospitalized patients from whom throat swabs were collected, including the five patients admitted to the ICU, tested positive for Ad14. Fifteen of these hospitalized patients tested negative for other respiratory pathogens, and one patient had a sputum culture that was positive for Haemophilus influenzae.
All health-care workers from hospital units where trainees had been admitted were offered testing for Ad14, regardless of history of respiratory illness. Of 218 health-care workers tested by PCR, six (3%) were positive for Ad14; five of the six reported direct contact with hospitalized Ad14 patients.
Prevention measures implemented during the outbreak included increasing the number of hand-sanitizing stations, widespread sanitizing of surfaces and equipment with appropriate disinfectants, increasing awareness of Ad14 among trainees and staff members, and taking contact and droplet precautions for hospitalized patients with Ad14. Beginning on May 26, trainees with febrile respiratory illness were confined to one dormitory and both patients and staff members were required to wear surgical masks.
Cases reported postinvestigation. Since the investigation, new cases of febrile respiratory illness have continued to occur at LAFB, but the weekly incidence has declined from a peak of 74 cases with onset during the week of May 27--June 2, to 55 cases with onset during the week of September 23--29 (the most recent period for which data were available). In addition, during March--September 2007, three other military bases in Texas that received trainees from LAFB reported a total of 220 cases of Ad14 illness (Air Force Institute for Operational Health, personal communication, 2007). However, whether Ad14 spread from LAFB to these three bases has not been determined. Ad14 also was detected in April in an eye culture from an outpatient in the surrounding community who had respiratory symptoms and conjunctivitis. No link between this case and the LAFB cases was identified.

Reported by: Oregon Dept of Human Svcs. Washington State Dept of Health Communicable Diseases. 37th Training Wing, 59th Hospital Wing, Air Force Institute for Operational Health, Epidemic and Outbreak Surveillance, US Air Force. Naval Health Research Center, US Navy. Texas Dept of State Health Svcs. New York City Dept of Health and Mental Hygiene. Div of Viral Diseases, National Center for Immunization and Respiratory Diseases; Div of Healthcare Quality Promotion, National Center for Preparedness, Detection, and Control of Infectious Diseases; Career Development Div, Office of Workforce and Career Development, CDC. Editorial Note:


Adenoviruses were first described in the 1950s and are associated with a broad spectrum of clinical illness, including conjunctivitis, febrile upper respiratory illness, pneumonia, and gastrointestinal disease. Severe illness can occur in newborn or elderly patients or in patients with underlying medical conditions but is generally not life-threatening in otherwise healthy adults. Adenoviruses are known to cause outbreaks of disease, including keratoconjunctivitis, and tracheobronchitis and other respiratory diseases among military recruits (2,3). Although adenovirus outbreaks in military recruits are well-recognized (3), infection usually does not require hospitalization and rarely requires admission to an ICU. Beyond the neonatal period, deaths associated with community-acquired adenovirus infection in persons who are not immunodeficient are uncommon and usually sporadic.
Fifty-one adenovirus serotypes have been identified (4). 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. Ad14 infection was described initially in 1955 (5) and was associated with epidemic acute respiratory disease in military recruits in Europe in 1969 (6) but has since been detected infrequently. For example, during 2001--2002, Ad14 was associated with approximately 8% of respiratory adenoviral infections in the pediatric ward of a Taiwan hospital, with approximately 40% of Ad14 cases in children aged 4--8 years manifesting as lower airway disease (7).
The National Surveillance for Emerging Adenovirus Infections system includes military and civilian laboratories at 15 sites. During 2004--2007, this surveillance system detected 17 isolates of Ad14 from seven sites (8). Ten of the 17 isolates (60%) were collected from three military bases (8). Despite this surveillance, adenovirus infections often go undetected, because few laboratories routinely test for adenovirus and even fewer do serotyping. Wider circulation of Ad14 might have occurred in recent years and might still be occurring.
Further work is needed to understand the natural history of Ad14, risk factors for severe Ad14 disease, and how Ad14 transmission can be prevented effectively. Vaccines against adenovirus serotypes four and seven (i.e., Ad4 and Ad7) were used among military recruits during 1971--1999, before vaccines were no longer available. Adenoviral disease among U.S. military recruits subsequently increased (9). Ad4 and Ad7 oral vaccines have been redeveloped and are being evaluated in clinical trials. Work is ongoing to determine whether the new Ad4 and Ad7 vaccines will protect against Ad14 infection. Management of adenoviral infections is largely supportive. A number of antiviral drugs, including ribavirin, vidarabine, and cidofovir, have been used to treat adenoviral infections such as Ad14, but none have shown definitive efficacy against adenoviruses (2).
Control of adenovirus outbreaks can be challenging because these viruses can be shed in both respiratory secretions and feces and can persist for weeks on environmental surfaces. Guidelines for the care of patients with pneumonia (10) should be followed in cases of suspected adenoviral pneumonia.

Clinicians with questions related to testing of patients for adenovirus or Ad14 infection should contact their state health departments, which can provide assistance. State health departments and military facilities should contact CDC to report unusual clusters of severe adenoviral disease or cases of Ad14 or to obtain additional information regarding laboratory testing. References</B>

1. Metzgar D, Osuna M, Kajon AE. Abrupt emergence of diverse species B1 and B2 adenoviruses in US military recruit training centers. J Infect Dis. In press.
2. Adenovirus. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious disease. 6th edition. Philadelphia, PA: Churchill Livingstone; 2004.
3. Dingle JH, Langmuir AD. Epidemiology of acute, respiratory disease in military recruits. Am Rev Respir Dis 1968;97(Suppl):1--65.
4. Kajon AE, Moseley JM, Metzgar D, et al. Molecular epidemiology of adenovirus type 4 infections in US military recruits in the postvaccination era (1997--2003). J Infect Dis 2007;196:67--75.
5. Van der Veen J, Kok G. Isolation and typing of adenoviruses recovered from military recruits with acute respiratory disease in The Netherlands. Am J Hyg 1957;65:119--29.
6. Hierholzer JC, Pumarola A. Antigenic characterization of intermediate adenovirus 14-11 strains associated with upper respiratory illness in a military camp. Infect Immun 1976;13:354--9.
7. Chen H, Chiou S, Hsiao H, et al. Respiratory adenoviral infections in children: a study of hospitalized cases in southern Taiwan in 2001--2002. J Trop Pediatr 2002;50:279--84.
8. National Surveillance for Emerging Adenovirus Infections. Available at http://www.public-health.uiowa.edu/adv.
9. Russell KL, Hawksworth AW, Ryan MA, et al. Vaccine-preventable adenoviral respiratory illness in US military recruits, 1999--2004. Vaccine 2006;24:2835--42.
10. CDC. Guidelines for preventing health-care--associated pneumonia, 2003. Recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee. MMWR 2004;53(No. RR-3).

<SMALL>* Defined as 1) fever >100.5?F (>38.1?C) plus at least one other sign or symptom of respiratory illness or 2) diagnosis of pneumonia.

</SMALL>Figure


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[Sally Furniss]

Re: CDC: New Respiratory Bug Has Killed 10

Lethal Cold Germ Becomes More Common, U.S. Health Experts Say

http://www.bloomberg.com/apps/news?pid=20601202&sid=ahWfH__NHsic&refer=healthcare

By Elizabeth Lopatto
<!-- WARNING: #foreach: $wnstory.ATTS: null at /bb/data/web/templates/webmacro_en/20601202.wm:289.2 --> <!-- WARNING: #foreach: $wnstory.ATTS: null at /bb/data/web/templates/webmacro_en/20601202.wm:303.19 --> Nov. 15 (Bloomberg) -- A cold germ turned deadly for 10 patients and sent dozens of others to the hospital, according to a U.S. health report that says infections with the severe virus strain are becoming more common.
The adenovirus, which usually causes mild cold symptoms, can cause severe lung disease in patients. The strain, called Ad14, sent fifty-three people to the hospital, including a 12- day-old infant, the Centers for Disease Control and Prevention said in its Morbidity and Mortality Weekly Report.
Controlling adenovirus outbreaks can be difficult because the germs are easily spread and can live for weeks on objects and surfaces, the agency said. These viruses aren't life- threatening to otherwise healthy adults, although newborn, elderly and patients with weakened immune systems can become seriously ill.
``Wider circulation of Ad14 might have occurred in recent years and might still be occurring,'' according to an editorial note accompanying the report.
Patients in New York, Oregon, Texas and Washington were identified as having the Ad14 strain, according to the report. The one case in New York resulted in the death of the 12-day-old infant in May 2006. In Oregon, 31 patients were identified with the virus; of these, six adults and one infant died from pneumonia. Those cases occurred between March and May this year.
Four cases were reported in a residential-care facility in Washington state. One patient, who already had AIDS, died. In Texas, 106 people at the Lackland Air Force Base were identified as carrying the virus, including 27 who were hospitalized. Five patients went to the intensive care unit, and one died.
No evidence exists that the outbreaks were related, the CDC said.
The Ad14 strain was first described in 1955, and caused respiratory disease in European military recruits. A number of antiviral drugs, including ribavirin, cidofovir and vidarabine, have been used to treat the illness. No drug has ``shown definitive efficacy against adenoviruses,'' the authors wrote.
To contact the reporter on this story: Elizabeth Lopatto in New York at elopatto@bloomberg.net .

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

The level of recombination in these isolates is remarkable.

[AlaskaDenise]

Re: CDC: New Respiratory Bug Has Killed 10

The level of recombination in these isolates is remarkable.

Are you saying there are a lot of changes, or that so much of those changes is due to recombination?

.

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

Are you saying there are a lot of changes, or that so much of those changes is due to recombination?

.

Adenovirus has OBVIOUS homologous recombination.

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

Are you saying there are a lot of changes, or that so much of those changes is due to recombination?

.

PCR analysis of egyptian respiratory adenovirus isolates, including identification of species, serotypes, and coinfections.

<!--AuthorList-->Metzgar D, Osuna M, Yingst S, Rakha M, Earhart K, Elyan D, Esmat H, Saad MD, Kajon A, Wu J, Gray GC, Ryan MA, Russell KL.
Naval Health Research Center, P.O. Box 85122, San Diego, CA 92186-5122, USA. metzgar@nhrc.navy.mil
Eighty-eight adenovirus (Ad) isolates and associated clinical data were collected from walk-in patients with influenza-like illness in Egypt during routine influenza surveillance from 1999 through 2002. Respiratory Ad distributions are geographically variable, and serotype prevalence has not been previously characterized in this region. Serotype identity is clinically relevant because it predicts vaccine efficacy and correlates strongly with both clinical presentation and epidemiological pattern. Species and serotype identities were determined using several well-validated multiplex PCR protocols culled from the literature and supplemented with a few novel primer sets designed to identify rare types. The isolates included common species B1 serotypes (Ad3 and Ad7), common species C serotypes (Ad1, Ad2, and Ad5), the less common species B2 serotype Ad11, and three isolates of the rare species B1 serotype Ad16. Two isolates that appear to be variant Ad16 were also identified. Fifteen coinfections of multiple adenoviral types, primarily AdB/AdC and Ad3/Ad7 dual infections, were detected. The majority of these were verified using redundant PCR tests targeted at multiple genes. PCR is able to resolve coinfections, in contrast to traditional serum neutralization tests. PCR is also comparatively rapid and requires very little equipment. Application of the method allowed an inclusive determination of the serotypes found in the Egyptian respiratory sample set and demonstrated that coinfections are common and may play a previously unrecognized role in adenovirus pathogenesis, evolution, and epidemiology. In particular, coinfections may influence adenoviral evolution, as interserotypic recombination has been identified as a source of emerging strains.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16272512&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

[AlaskaDenise]

Re: CDC: New Respiratory Bug Has Killed 10

Interesting that out of 88 cases, 15 had coinfections!

and...

.....coinfections are common and may play a previously unrecognized role in adenovirus pathogenesis, evolution, and epidemiology. In particular, coinfections may influence adenoviral evolution.....

.

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

Interesting that out of 88 cases, 15 had coinfections!

and...



.

Yes, recombination requires co-infections, which are common for adenovirus and influenza.

Recombination is the name of the game.

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

The Journal of Infectious Diseases 2007;196:1465-1473
This article is in the public domain, and no copyright is claimed.
0022-1899/2007/19610-0008
<HR><TABLE cellPadding=5><TBODY><TR><TD align=middle bgColor=#990000>MAJOR<SUP> </SUP>ARTICLE</TD><SUP></SUP></TR></TBODY></TABLE>

Abrupt<SUP> </SUP>Emergence<SUP> </SUP>of<SUP> </SUP>Diverse<SUP> </SUP>Species<SUP> </SUP>B<SUP> </SUP>Adenoviruses<SUP> </SUP>at<SUP> </SUP>US<SUP> </SUP>Military<SUP> </SUP>Recruit<SUP> </SUP>Training<SUP> </SUP>Centers<SUP></SUP><SUP></SUP><SUP> </SUP>

David Metzgar,<SUP>1</SUP><SUP> </SUP>Miguel Osuna,<SUP>1</SUP><SUP> </SUP>Adriana E. Kajon,<SUP>2</SUP><SUP> </SUP>Anthony W. Hawksworth,<SUP>1</SUP><SUP> </SUP>Marina Irvine,<SUP>1</SUP><SUP> </SUP>and<SUP> </SUP>Kevin L. Russell<SUP>1</SUP><SUP> </SUP><SUP>1</SUP>Respiratory<SUP> </SUP>Disease<SUP> </SUP>Laboratory,<SUP> </SUP>Department<SUP> </SUP>of<SUP> </SUP>Defense<SUP> </SUP>Center<SUP> </SUP>for<SUP> </SUP>Deployment<SUP> </SUP>Health<SUP> </SUP>Research,<SUP> </SUP>Naval<SUP> </SUP>Health<SUP> </SUP>Research<SUP> </SUP>Center,<SUP> </SUP>San<SUP> </SUP>Diego,<SUP> </SUP>California;<SUP> </SUP><SUP>2</SUP>Infectious<SUP> </SUP>Disease<SUP> </SUP>Program,<SUP> </SUP>Lovelace<SUP> </SUP>Respiratory<SUP> </SUP>Research<SUP> </SUP>Institute,<SUP> </SUP>Albuquerque,<SUP> </SUP>New<SUP> </SUP>Mexico<SUP> </SUP>

<SUP></SUP></B>
<CENTER><TABLE cellSpacing=0 cellPadding=0 width="80%" border=0><TBODY><TR><TD>
Background. Adenoviruses (Ads) cause continuous<SUP> </SUP>outbreaks of acute respiratory<SUP> </SUP>disease (ARD) in US<SUP> </SUP>military training facilities. In<SUP> </SUP>1996, the loss of<SUP> </SUP>vaccines targeting the dominant<SUP> </SUP>recruit-associated serotypes precipitated the<SUP> </SUP>reemergence of Ads in<SUP> </SUP>these populations. Between 1999<SUP> </SUP>and 2002, serotype 4<SUP> </SUP>accounted for >95% of<SUP> </SUP>Ads isolated from recruits<SUP> </SUP>and for >50% of<SUP> </SUP>ARD cases in training<SUP> </SUP>facilities (15,000 cases/year).
Methods. Ads (<!--Start Italic-->n<!--End Italic--><SUP> </SUP>= 1867) collected between<SUP> </SUP>2002 and 2006 from<SUP> </SUP>recruits with ARD at<SUP> </SUP>8 military training facilities<SUP> </SUP>in the United States<SUP> </SUP>were serotyped by serum<SUP> </SUP>neutralization and polymerase chain<SUP> </SUP>reaction.<SUP> </SUP>
Results. The dominance of Ad4<SUP> </SUP>continued through 2005, followed<SUP> </SUP>by a simultaneous emergence<SUP> </SUP>of diverse species B<SUP> </SUP>serotypes at the majority<SUP> </SUP>of sites. This included<SUP> </SUP>the subspecies B1 serotypes<SUP> </SUP>3, 7, and 21<SUP> </SUP>and the subspecies B2<SUP> </SUP>serotype 14. Ad14 was<SUP> </SUP>the most prevalent species<SUP> </SUP>B serotype, appearing in<SUP> </SUP>high numbers at 3<SUP> </SUP>sites and becoming dominant<SUP> </SUP>at 1.<SUP> </SUP>
Conclusions. Subspecies B2 Ads<SUP> </SUP>have rarely been associated<SUP> </SUP>with ARD, and only<SUP> </SUP>in Eurasia. This survey<SUP> </SUP>represents the first report<SUP> </SUP>of AdB2-associated ARD in<SUP> </SUP>the Western Hemisphere. The<SUP> </SUP>simultaneous emergence of several<SUP> </SUP>species B Ads suggests<SUP> </SUP>a common external source<SUP> </SUP>(the civilian population) and<SUP> </SUP>a decrease in preexisting<SUP> </SUP>immunity to species B<SUP> </SUP>Ads.<SUP> </SUP>

</TD></TR></TBODY></TABLE></CENTER><HR>
Received 17 April 2007; accepted 11 May 2007; electronically published 31 October 2007.<SUP> </SUP>
Potential conflicts of interest:<SUP> </SUP>none reported.<SUP> </SUP>
Financial support: Department<SUP> </SUP>of Defense Global Emerging<SUP> </SUP>Infections Surveillance and Response<SUP> </SUP>System under research work<SUP> </SUP>unit 60701 (Naval Health<SUP> </SUP>Research Center report 07-12).<SUP> </SUP>
The<SUP> </SUP>views expressed in this<SUP> </SUP>work are those of<SUP> </SUP>the authors and do<SUP> </SUP>not reflect the official<SUP> </SUP>policy or position of<SUP> </SUP>the Department of the<SUP> </SUP>Navy, Department of the<SUP> </SUP>Army, the Department of<SUP> </SUP>Defense, or the US<SUP> </SUP>government.<SUP> </SUP>
(See the editorial commentary<SUP> </SUP>by Binn et al., on pages<SUP> </SUP>1436?7.)<SUP> </SUP>
Reprints<SUP> </SUP>or<SUP> </SUP>correspondence:<SUP> </SUP>Dr.<SUP> </SUP>David<SUP> </SUP>Metzgar,<SUP> </SUP>Naval<SUP> </SUP>Health<SUP> </SUP>Research<SUP> </SUP>Center,<SUP> </SUP>PO<SUP> </SUP>Box<SUP> </SUP>85122,<SUP> </SUP>San<SUP> </SUP>Diego,<SUP> </SUP>CA<SUP> </SUP>92186<SUP> </SUP>(david.metzgar@med.navy.mil).


<HR> Adenoviruses (Ads) were discovered<SUP> </SUP>in 1953 [1]. The<SUP> </SUP>diverse human pathogens in<SUP> </SUP>this family are categorized<SUP> </SUP>by hemagglutination into species<SUP> </SUP>A?F. They are further<SUP> </SUP>characterized by serum neutralization,<SUP> </SUP>and there are 51<SUP> </SUP>currently recognized serotypes (Ad1?51)<SUP> </SUP>[2?4]. Higher resolution is<SUP> </SUP>offered by whole-genome restriction<SUP> </SUP>enzyme analysis (genome typing)<SUP> </SUP>[5]. Ad genomes are<SUP> </SUP>stable and recombinations are<SUP> </SUP>rare, allowing inference of<SUP> </SUP>species and serotype from<SUP> </SUP>single-locus polymerase chain reaction<SUP> </SUP>(PCR) or sequence data<SUP> </SUP>[6?9].<SUP> </SUP>
Human Ads of species<SUP> </SUP>B (specifically, subspecies B1),<SUP> </SUP>C, and E are<SUP> </SUP>globally distributed pathogens responsible<SUP> </SUP>for outbreaks of respiratory<SUP> </SUP>disease and conjunctivitis [10,<SUP> </SUP>11]. Species C Ads<SUP> </SUP>are ubiquitous and endemic<SUP> </SUP>and are primarily responsible<SUP> </SUP>for childhood illness. Healthy<SUP> </SUP>adults are more likely<SUP> </SUP>to suffer from respiratory<SUP> </SUP>disease caused by AdE<SUP> </SUP>and AdB1 [10], against<SUP> </SUP>which a larger proportion<SUP> </SUP>of adults lack protective<SUP> </SUP>antibodies [11, 12]. Subspecies<SUP> </SUP>B1 Ads, most commonly<SUP> </SUP>serotypes 3 and 7,<SUP> </SUP>cause recurring epidemics of<SUP> </SUP>febrile acute respiratory disease<SUP> </SUP>(ARD) in civilian populations,<SUP> </SUP>with symptoms ranging from<SUP> </SUP>influenza-like fever and discomfort<SUP> </SUP>to pneumonia and death<SUP> </SUP>[6, 10, 11]. These<SUP> </SUP>serotypes, along with the<SUP> </SUP>AdB1 serotype 21 and<SUP> </SUP>the AdE serotype 4,<SUP> </SUP>are also responsible for<SUP> </SUP>essentially continuous epidemics of<SUP> </SUP>ARD at military recruit<SUP> </SUP>training facilities worldwide [12?15].<SUP> </SUP>Prevaccination data from US<SUP> </SUP>military training facilities showed<SUP> </SUP>that 20% of all<SUP> </SUP>trainees had to be<SUP> </SUP>removed from training for<SUP> </SUP>a week or more<SUP> </SUP>for recovery from Ad-associated<SUP> </SUP>illness, 40% became ill,<SUP> </SUP>and 80% were infected<SUP> </SUP>[16]. Current data, collected<SUP> </SUP>since the loss of<SUP> </SUP>the Ad4 and Ad7<SUP> </SUP>vaccines previously used by<SUP> </SUP>the US military, reflect<SUP> </SUP>similar trends (6.4% removed<SUP> </SUP>from training, 25% ill,<SUP> </SUP>98% of those initially<SUP> </SUP>seronegative seroconverted) [17]. US<SUP> </SUP>recruits suffer 22,000 cases<SUP> </SUP>of reported febrile ARD<SUP> </SUP>per year, 15,000 of<SUP> </SUP>which are associated with<SUP> </SUP>Ads [18]. Many more<SUP> </SUP>Ad infections go unreported<SUP> </SUP>in this population. Studies<SUP> </SUP>have shown that less<SUP> </SUP>than half of recruits<SUP> </SUP>with ARD seek medical<SUP> </SUP>care [17, 18].<SUP> </SUP>
Between 1971<SUP> </SUP>and 1996, the United<SUP> </SUP>States used Ad4 and<SUP> </SUP>Ad7 vaccines to successfully<SUP> </SUP>control adenoviral illness in<SUP> </SUP>recruit populations, decreasing ARD<SUP> </SUP>by 50%?60% and decreasing<SUP> </SUP>Ad-associated ARD by >95%<SUP> </SUP>[11, 13, 18]. Other<SUP> </SUP>subspecies B1 Ads, including<SUP> </SUP>Ad3 and Ad21, appeared<SUP> </SUP>in vaccinated recruit populations,<SUP> </SUP>although they did not<SUP> </SUP>cause a return to<SUP> </SUP>prevaccination levels of ARD<SUP> </SUP>[14, 19, 20].<SUP> </SUP>
Subspecies B2<SUP> </SUP>Ads are generally associated<SUP> </SUP>with sporadic diseases of<SUP> </SUP>the kidney and urinary<SUP> </SUP>tract [3]. The literature<SUP> </SUP>has, however, recorded occasional<SUP> </SUP>respiratory disease outbreaks associated<SUP> </SUP>with 2 AdB2 serotypes,<SUP> </SUP>Ad11 and Ad14 [21,<SUP> </SUP>22].<SUP> </SUP>
The US military has<SUP> </SUP>conducted population-based ARD surveillance<SUP> </SUP>at 8 US training<SUP> </SUP>facilities since the loss<SUP> </SUP>of vaccine production in<SUP> </SUP>1996, through the subsequent<SUP> </SUP>gradual reduction in vaccination<SUP> </SUP>between 1997 and 1999,<SUP> </SUP>and through the following<SUP> </SUP>period of no vaccination<SUP> </SUP>[13, 18, 20]. We<SUP> </SUP>present here data describing<SUP> </SUP>the serotype distributions at<SUP> </SUP>those facilities during the<SUP> </SUP>years 2002?2006. As part<SUP> </SUP>of ongoing population-based surveillance<SUP> </SUP>at these sites, 14,548<SUP> </SUP>samples were collected and<SUP> </SUP>tested for Ad. The<SUP> </SUP>resulting numbers, paired with<SUP> </SUP>data on population size,<SUP> </SUP>were used to generate<SUP> </SUP>estimates of Ad-associated ARD<SUP> </SUP>rates. From the resulting<SUP> </SUP>set of cultured isolates,<SUP> </SUP>1867 samples were randomly<SUP> </SUP>chosen and typed by<SUP> </SUP>either serotype-specific antibody neutralization<SUP> </SUP>(through March 2004) or<SUP> </SUP>serotype-specific PCR (beginning in<SUP> </SUP>April 2004; a PCR<SUP> </SUP>flow chart is shown<SUP> </SUP>in figure 1). The resulting<SUP> </SUP>serotype identifications were verified<SUP> </SUP>in a subset of<SUP> </SUP>cases by sequencing the<SUP> </SUP>primary antigenic determinant [8]<SUP> </SUP>and by genome typing<SUP> </SUP>[5].<SUP> </SUP>
<TABLE cellSpacing=10><TBODY><TR><TD vAlign=top align=middle>
(49 kB)</TD><TD vAlign=top align=left>Figure 1. Flow chart representing the<SUP> </SUP>series of polymerase chain<SUP> </SUP>reactions (PCRs) used to<SUP> </SUP>identify adenovirus (Ad) strains<SUP> </SUP>in this study. The<SUP> </SUP>PCR indicated by the<SUP> </SUP>asterisks discriminates the highly<SUP> </SUP>divergent genome types Ad4a<SUP> </SUP>and Ad4p on the<SUP> </SUP>basis of an amplicon<SUP> </SUP>length polymorphism (see table 1).<SUP> </SUP>ARD, acute respiratory disease.
</TD></TR></TBODY></TABLE>
To<SUP> </SUP>address the epidemiological consequences<SUP> </SUP>of serotype turnover events,<SUP> </SUP>rate data are presented<SUP> </SUP>for 1 site at<SUP> </SUP>which a particularly sharp<SUP> </SUP>serotype emergence occurred. Serotyping<SUP> </SUP>results are discussed in<SUP> </SUP>relation to both general<SUP> </SUP>Ad epidemiology and in<SUP> </SUP>relation to the current<SUP> </SUP>effort to reinstitute the<SUP> </SUP>Ad4 and Ad7 vaccines.<SUP> </SUP>
MATERIALS AND METHODS<SUP> </SUP>
ARD case definition and sample collection. Collaborators<SUP> </SUP>and Naval Health Research<SUP> </SUP>Center (NHRC) staff at<SUP> </SUP>each site monitor trainee<SUP> </SUP>populations for symptoms of<SUP> </SUP>febrile respiratory illness. A<SUP> </SUP>case of febrile respiratory<SUP> </SUP>illness is defined as<SUP> </SUP>occurring when a recruit<SUP> </SUP>presents for medical care<SUP> </SUP>and meets the following<SUP> </SUP>2 criteria: fever with<SUP> </SUP>oral temperature 38?C (100.5?F)<SUP> </SUP>and a respiratory symptom<SUP> </SUP>(cough or sore throat).<SUP> </SUP>Clinical specimens are obtained<SUP> </SUP>as pharyngeal swabs in<SUP> </SUP>viral transport medium (Remel)<SUP> </SUP>from a subset of<SUP> </SUP>recruits who seek medical<SUP> </SUP>care. These samples are<SUP> </SUP>then sent on dry<SUP> </SUP>ice to the NHRC<SUP> </SUP>to be tested for<SUP> </SUP>respiratory pathogens.<SUP> </SUP>
Origin of virus strains. Serotyping work was<SUP> </SUP>done on a collection<SUP> </SUP>of 1867 Ad strains<SUP> </SUP>isolated from the pharyngeal<SUP> </SUP>swabs of military trainees<SUP> </SUP>presenting with symptoms of<SUP> </SUP>ARD between 2002 and<SUP> </SUP>2006 at 8 training<SUP> </SUP>sites in the Unites<SUP> </SUP>States: Fort Jackson, South<SUP> </SUP>Carolina; Fort Benning, Georgia;<SUP> </SUP>Fort Leonard Wood, Missouri<SUP> </SUP>(all Army); Naval Recruit<SUP> </SUP>Training Command, Great Lakes,<SUP> </SUP>Illinois; Lackland Air Force<SUP> </SUP>Base, San Antonio, Texas;<SUP> </SUP>Coast Guard Training Center,<SUP> </SUP>Cape May, New Jersey;<SUP> </SUP>Marine Corps Recruit Depot<SUP> </SUP>(MCRD) Parris Island, Parris<SUP> </SUP>Island, South Carolina; and<SUP> </SUP>MCRD San Diego, San<SUP> </SUP>Diego, California. Initial identifications<SUP> </SUP>were made in the<SUP> </SUP>College of American Pathologists?accredited<SUP> </SUP>NHRC diagnostic microbiology laboratory<SUP> </SUP>either by culture/immunofluorescence in<SUP> </SUP>A549 cell monolayers or<SUP> </SUP>by PCR. PCR identifications<SUP> </SUP>were followed by culture/immunofluorescence<SUP> </SUP>to verify the result<SUP> </SUP>and produce high-titer stocks<SUP> </SUP>for molecular serotype analysis.<SUP> </SUP>
Serotype determination. The<SUP> </SUP>serotype identifications presented in<SUP> </SUP>this study were done<SUP> </SUP>using a microneutralization assay<SUP> </SUP>[23], PCR, or both.<SUP> </SUP>PCR primers, as well<SUP> </SUP>as the source of<SUP> </SUP>the primers and references<SUP> </SUP>to previous validations in<SUP> </SUP>the hands of the<SUP> </SUP>original authors, are shown<SUP> </SUP>in table 1. PCR methods<SUP> </SUP>have been used as<SUP> </SUP>a primary means of<SUP> </SUP>serotype identification since April<SUP> </SUP>2004, when type-specific serum<SUP> </SUP>became increasingly difficult to<SUP> </SUP>obtain [6]. These methods<SUP> </SUP>have also been retrospectively<SUP> </SUP>applied to thousands of<SUP> </SUP>archived samples collected before<SUP> </SUP>2003, which were initially<SUP> </SUP>serotyped by traditional neutralization<SUP> </SUP>(by both ourselves [6,<SUP> </SUP>20, and unpublished data]<SUP> </SUP>and others [7, 9])<SUP> </SUP>to validate the ability<SUP> </SUP>of PCR to predict<SUP> </SUP>serotype. All such validations<SUP> </SUP>have shown an exceptionally<SUP> </SUP>high level of correlation<SUP> </SUP>between different methods.<SUP> </SUP>
<TABLE cellSpacing=10><TBODY><TR><TD vAlign=top align=middle></TD><TD vAlign=top align=left>Table 1. Primers.</TD></TR></TBODY></TABLE>
We performed<SUP> </SUP>a validation of PCR<SUP> </SUP>relative to serum neutralization<SUP> </SUP>by using PCR to<SUP> </SUP>reanalyze isolates previously serotyped<SUP> </SUP>by neutralization, including all<SUP> </SUP>serotypes that had been<SUP> </SUP>collected by the laboratory<SUP> </SUP>during the previous 5<SUP> </SUP>years of recruit surveillance<SUP> </SUP>activity. Initially, PCR identifications<SUP> </SUP>of species B and<SUP> </SUP>E were made using<SUP> </SUP>the B/E portion of<SUP> </SUP>the A?F species multiplex<SUP> </SUP>[9], as described elsewhere<SUP> </SUP>[6]. Later, we developed<SUP> </SUP>the BCE multiplex (described<SUP> </SUP>below) in order to<SUP> </SUP>capture all respiratory species<SUP> </SUP>(B, C, and E)<SUP> </SUP>while simultaneously differentiating the<SUP> </SUP>2 very distinct genome<SUP> </SUP>types of Ad4, 4a<SUP> </SUP>and 4p [24]. Both<SUP> </SUP>methods were validated using<SUP> </SUP>the same set of<SUP> </SUP>cultured clinical specimens, and<SUP> </SUP>both yielded identical results.<SUP> </SUP>Results are shown only<SUP> </SUP>for the BCE multiplex.<SUP> </SUP>A similar validation was<SUP> </SUP>performed for the published<SUP> </SUP>Ad3/Ad7/Ad21 serotype-specific multiplex [7]<SUP> </SUP>used in this study.<SUP> </SUP>The numbers of clinical<SUP> </SUP>isolates used in each<SUP> </SUP>validation, along with the<SUP> </SUP>resulting sensitivity and specificity<SUP> </SUP>measures, are shown in<SUP> </SUP>table 2.<SUP> </SUP>
<TABLE cellSpacing=10><TBODY><TR><TD vAlign=top align=middle></TD><TD vAlign=top align=left>Table 2. Performance of polymerase chain<SUP> </SUP>reaction (PCR)?based adenovirus (Ad)<SUP> </SUP>identification, species typing, and<SUP> </SUP>serotyping with respect to<SUP> </SUP>culture/immunofluorescence and serum neutralization.</TD></TR></TBODY></TABLE>
The<SUP> </SUP>primers used to test<SUP> </SUP>for Ad11 and Ad14<SUP> </SUP>[6] were developed in-house<SUP> </SUP>using all available sequence<SUP> </SUP>data and tested for<SUP> </SUP>sensitivity against appropriate control<SUP> </SUP>strains from the American<SUP> </SUP>Type Culture Collection. These<SUP> </SUP>serotypes had never been<SUP> </SUP>seen before in US<SUP> </SUP>recruit ARD surveillance; therefore,<SUP> </SUP>clinical samples previously identified<SUP> </SUP>as Ad11 or Ad14<SUP> </SUP>were unavailable for validation.<SUP> </SUP>Positive identifications made using<SUP> </SUP>these primers were verified<SUP> </SUP>by secondary analysis of<SUP> </SUP>a subset of samples,<SUP> </SUP>including 1 each from<SUP> </SUP>San Diego, Great Lakes,<SUP> </SUP>and Fort Benning, by<SUP> </SUP>sequencing hypervariable region (HVR)<SUP> </SUP>7 of the hexon<SUP> </SUP>gene [8]. BLAST analysis<SUP> </SUP>[25] of these sequences<SUP> </SUP>by means of GenBank<SUP> </SUP>allowed sequence-based assignment of<SUP> </SUP>serotype. The analyzed gene<SUP> </SUP>includes the primary serotype<SUP> </SUP>determinants and, hence, offers<SUP> </SUP>the most direct opportunity<SUP> </SUP>for molecular inference of<SUP> </SUP>serotype [8].<SUP> </SUP>
All PCRs were<SUP> </SUP>subjected to specificity tests<SUP> </SUP>against control samples of<SUP> </SUP>a wide variety of<SUP> </SUP>respiratory pathogens, commensals, and<SUP> </SUP>environmentally common bacteria (40<SUP> </SUP>bacteria and 50 viruses),<SUP> </SUP>including several serotypes of<SUP> </SUP>Ad. No cross-reactions were<SUP> </SUP>detected (data not shown).<SUP> </SUP>Specificity testing also included<SUP> </SUP>a large number of<SUP> </SUP>culture-negative patient specimens, as<SUP> </SUP>indicated in table 2.<SUP> </SUP>
Secondary verification<SUP> </SUP>of the serotype of<SUP> </SUP>all Ad3, Ad7, Ad14,<SUP> </SUP>and Ad21 isolates and<SUP> </SUP>>100 Ad4 isolates was<SUP> </SUP>performed at the Lovelace<SUP> </SUP>Respiratory Research Institute by<SUP> </SUP>traditional whole-genome restriction enzyme<SUP> </SUP>analysis with <!--Start Italic-->Bam<!--End Italic-->HI (genome<SUP> </SUP>typing) [24].<SUP> </SUP>
PCR. PCRs were done<SUP> </SUP>as shown in the<SUP> </SUP>flow chart (figure 1). Both<SUP> </SUP>monoplex and multiplex PCRs<SUP> </SUP>were formulated as described<SUP> </SUP>elsewhere [6], under essentially<SUP> </SUP>standard conditions with the<SUP> </SUP>addition of the solvent<SUP> </SUP>betaine (Q-Solution; Qiagen). The<SUP> </SUP>only exception to the<SUP> </SUP>previously described conditions was<SUP> </SUP>for the BCE multiplex,<SUP> </SUP>which used 0.1 mol/L<SUP> </SUP>concentration of each primer<SUP> </SUP>instead of 0.2 mol/L.<SUP> </SUP>
Cycling<SUP> </SUP>conditions included a 10-min<SUP> </SUP>final extension at 72?C<SUP> </SUP>and a final hold<SUP> </SUP>at 4?C. Regarding other<SUP> </SUP>parameters, reactions were cycled<SUP> </SUP>as shown in table 3.<SUP> </SUP>
<TABLE cellSpacing=10><TBODY><TR><TD vAlign=top align=middle></TD><TD vAlign=top align=left>Table 3. Polymerase<SUP> </SUP>chain reaction (PCR) conditions.</TD></TR></TBODY></TABLE>
All<SUP> </SUP>products were analyzed by<SUP> </SUP>standard agarose gel electrophoresis<SUP> </SUP>and ethidium bromide/UV visualization,<SUP> </SUP>as described elsewhere [6].<SUP> </SUP>
Development of a new PCR test for respiratory Ads. The<SUP> </SUP>novel BCE PCR test<SUP> </SUP>used in the present<SUP> </SUP>work was developed to<SUP> </SUP>target the relevant species<SUP> </SUP>(B, C, and E)<SUP> </SUP>by use of a<SUP> </SUP>gene (E1A) that is<SUP> </SUP>very well conserved within<SUP> </SUP>species but that is<SUP> </SUP>highly divergent between species<SUP> </SUP>while simultaneously discriminating between<SUP> </SUP>the 2 primary genome<SUP> </SUP>types of species E,<SUP> </SUP>Ad4a and Ad4p. Discrimination<SUP> </SUP>of Ad4a and Ad4p<SUP> </SUP>is based on a<SUP> </SUP>30-bp insertion/deletion mutation in<SUP> </SUP>the E1A gene. This<SUP> </SUP>PCR was briefly described<SUP> </SUP>in an earlier work<SUP> </SUP>[24], in which it<SUP> </SUP>was used to confirm<SUP> </SUP>the results of another<SUP> </SUP>test.<SUP> </SUP>
Sequence analysis of hexon genes. Fragments of 599 bp<SUP> </SUP>spanning HVR7 of the<SUP> </SUP>hexon gene of Ad14<SUP> </SUP>were amplified and sequenced<SUP> </SUP>using the primers HVR7f<SUP> </SUP>(5-GTCTTATGTACTATAACAGTACTGG-3) and HVR7r (5-GTGGTTGAATGGGTTGAC-3).<SUP> </SUP>Amplification reactions contained 1.5<SUP> </SUP>mmol/L MgCl2, 1 U<SUP> </SUP>of iProof <!--Start Italic-->Taq<!--End Italic--> polymerase<SUP> </SUP>(Bio-Rad Laboratories), 200 mol/L<SUP> </SUP>dNTP, 1? iProof high-fidelity<SUP> </SUP>buffer, and 1 mol/L<SUP> </SUP>each primer in a<SUP> </SUP>total volume of 50<SUP> </SUP>L. Cycling conditions consisted<SUP> </SUP>of denaturation for 30<SUP> </SUP>s at 94?C followed<SUP> </SUP>by 50 cycles of<SUP> </SUP>30 s at 94?C,<SUP> </SUP>45 s at 51?C,<SUP> </SUP>and 45 s at<SUP> </SUP>72?C. A final extension<SUP> </SUP>for 5 min at<SUP> </SUP>72?C was added. Amplicons<SUP> </SUP>were purified with Montage<SUP> </SUP>PCR columns and Micropure-EZ<SUP> </SUP>columns (Millipore). Sequencing reactions<SUP> </SUP>used the same primers<SUP> </SUP>and standard conditions. Sequencing<SUP> </SUP>services were contracted from<SUP> </SUP>the DNA Research Services<SUP> </SUP>of the University of<SUP> </SUP>New Mexico Health Sciences<SUP> </SUP>Center.<SUP> </SUP>
Sequence data analysis was<SUP> </SUP>done using SeqMan software<SUP> </SUP>for contig assembly and<SUP> </SUP>MegAlign software for sequence<SUP> </SUP>alignments (Lasergene 7.0.0; DNAStar).<SUP> </SUP>Ad14 hexon sequences were<SUP> </SUP>deposited in GenBank under<SUP> </SUP>accession numbers EF503568, EF503569,<SUP> </SUP>and EF503570.<SUP> </SUP>
Ethics. This research has<SUP> </SUP>been conducted in compliance<SUP> </SUP>with all applicable federal<SUP> </SUP>and international regulations governing<SUP> </SUP>the protection of human<SUP> </SUP>subjects in research (Department<SUP> </SUP>of Defense protocol NHRC.2005.0017).<SUP> </SUP>
RESULTS<SUP> </SUP>
Serotype<SUP> </SUP>data are shown in<SUP> </SUP>figure 2. Ad4 (species E)<SUP> </SUP>was identified in >95%<SUP> </SUP>of recruit ARD samples<SUP> </SUP>collected from 2002 through<SUP> </SUP>2005, following the trend<SUP> </SUP>seen in the preceding<SUP> </SUP>surveillance period [20]. Ad3,<SUP> </SUP>Ad7, and Ad21 (all<SUP> </SUP>subspecies B1) were occasionally<SUP> </SUP>seen during this time<SUP> </SUP>period, as were 3<SUP> </SUP>scattered cases of species<SUP> </SUP>C Ads. Ad3 cases<SUP> </SUP>were concentrated at San<SUP> </SUP>Diego and Fort Benning<SUP> </SUP>in 2004. Ad7 cases<SUP> </SUP>were clustered at Great<SUP> </SUP>Lakes in 2004, and<SUP> </SUP>Ad21 was primarily identified<SUP> </SUP>at Parris Island in<SUP> </SUP>2005.<SUP> </SUP>
<TABLE cellSpacing=10><TBODY><TR><TD vAlign=top align=middle>
(120 kB)</TD><TD vAlign=top align=left>Figure 2. Serotype identification of 1867<SUP> </SUP>adenovirus (Ad) isolates from<SUP> </SUP>military recruits at 8<SUP> </SUP>US training centers, 2002?2006.<SUP> </SUP>Each block represents 1<SUP> </SUP>sample from a recruit<SUP> </SUP>with febrile acute respiratory<SUP> </SUP>disease. Split blocks represent<SUP> </SUP>coinfections with 2 serotypes.<SUP> </SUP>The sample to the<SUP> </SUP>right of the asterisk<SUP> </SUP>was a triple infection<SUP> </SUP>with Ad4, Ad7, and<SUP> </SUP>Ad14. The decrease in<SUP> </SUP>sampling density at Lackland<SUP> </SUP>after 2004 resulted from<SUP> </SUP>a decrease in trainee<SUP> </SUP>density that essentially ended<SUP> </SUP>Ad outbreaks at that<SUP> </SUP>site. Otherwise, changes in<SUP> </SUP>sample volume are not<SUP> </SUP>representative of changes in<SUP> </SUP>disease rate but rather<SUP> </SUP>of changes in sampling<SUP> </SUP>effort. The relative proportions<SUP> </SUP>of different serotypes are<SUP> </SUP>representative of the relative<SUP> </SUP>frequency of isolation: Ad-positive<SUP> </SUP>samples were chosen randomly<SUP> </SUP>for serotyping. Results before<SUP> </SUP>April 2004 are from<SUP> </SUP>traditional serum neutralization; later<SUP> </SUP>results are from type-specific<SUP> </SUP>polymerase chain reaction.
</TD></TR></TBODY></TABLE>
In March<SUP> </SUP>and April of 2006,<SUP> </SUP>Ad14 (subspecies B2) simultaneously<SUP> </SUP>emerged at 5 training<SUP> </SUP>centers, including San Diego,<SUP> </SUP>Lackland, Fort Leonard Wood,<SUP> </SUP>Great Lakes, and Fort<SUP> </SUP>Benning, thereby affecting all<SUP> </SUP>services except the Coast<SUP> </SUP>Guard (Cape May). At<SUP> </SUP>3 of these centers,<SUP> </SUP>Ad14 first appeared in<SUP> </SUP>coinfections with AdB1 serotypes<SUP> </SUP>and in 1 case<SUP> </SUP>as part of a<SUP> </SUP>triple infection with Ad4<SUP> </SUP>and Ad7. Ad14 maintained<SUP> </SUP>a consistent presence at<SUP> </SUP>Fort Benning, San Diego,<SUP> </SUP>and Great Lakes during<SUP> </SUP>summer 2006. At the<SUP> </SUP>same time, a variety<SUP> </SUP>of AdB1 serotypes emerged<SUP> </SUP>at all sites except<SUP> </SUP>Cape May. Ad3 cases<SUP> </SUP>were clustered at Fort<SUP> </SUP>Jackson, Ad7 at Fort<SUP> </SUP>Leonard Wood and Parris<SUP> </SUP>Island, and Ad21 at<SUP> </SUP>Parris Island and Fort<SUP> </SUP>Benning. All 6 identified<SUP> </SUP>serotypes were seen at<SUP> </SUP>Fort Benning during one<SUP> </SUP>2-month period. Many coinfections<SUP> </SUP>were identified, the majority<SUP> </SUP>of which were coinfections<SUP> </SUP>with Ad4 and one<SUP> </SUP>of the species B<SUP> </SUP>serotypes.<SUP> </SUP>
All whole-genome <!--Start Italic-->Bam<!--End Italic-->HI restriction<SUP> </SUP>patterns (genome types) were<SUP> </SUP>concordant with PCR-inferred serotypes<SUP> </SUP>(or subspecies type, in<SUP> </SUP>the case of many<SUP> </SUP>of the AdB serotypes?<!--Start Italic-->Bam<!--End Italic-->HI<SUP> </SUP>patterns can readily distinguish<SUP> </SUP>B1 from B2 subspecies,<SUP> </SUP>but many serotype determinations<SUP> </SUP>require analysis with additional<SUP> </SUP>enzymes). Likewise, all identifications<SUP> </SUP>based on hexon sequencing<SUP> </SUP>were concordant with the<SUP> </SUP>original PCR-inferred identifications. ARD<SUP> </SUP>rates at MCRD San<SUP> </SUP>Diego, the site with<SUP> </SUP>the most complete turnover<SUP> </SUP>between Ad4 and Ad14<SUP> </SUP>(i.e., between species E<SUP> </SUP>and subspecies B2), did<SUP> </SUP>not shift noticeably during<SUP> </SUP>the most distinct period<SUP> </SUP>of Ad14 dominance (figure 3).<SUP> </SUP>Note that the general<SUP> </SUP>increase in the isolation<SUP> </SUP>rate over time is<SUP> </SUP>not a function of<SUP> </SUP>increased disease burden but<SUP> </SUP>rather of increased sampling<SUP> </SUP>effort.<SUP> </SUP>
<TABLE cellSpacing=10><TBODY><TR><TD vAlign=top align=middle>
(51 kB)</TD><TD vAlign=top align=left>Figure 3. Acute respiratory disease (ARD)<SUP> </SUP>rate at the Marine<SUP> </SUP>Corps Recruit Depot (MCRD)<SUP> </SUP>San Diego, 2002?2006. The<SUP> </SUP>red line indicates the<SUP> </SUP>approximate time at which<SUP> </SUP>adenovirus (Ad) 14 emerged<SUP> </SUP>as a significant contributor<SUP> </SUP>to the Ad burden<SUP> </SUP>at this site. The<SUP> </SUP>figure suggests that the<SUP> </SUP>emergence of Ad14 did<SUP> </SUP>not cause a greatly<SUP> </SUP>increased level of ARD<SUP> </SUP>but rather that Ad14<SUP> </SUP>partially replaced Ad4 as<SUP> </SUP>a causative agent, with<SUP> </SUP>little impact on the<SUP> </SUP>overall rate of ARD.
</TD></TR></TBODY></TABLE>
DISCUSSION<SUP> </SUP>
All<SUP> </SUP>AdB1 serotypes previously seen<SUP> </SUP>in recruit training centers<SUP> </SUP>in the United States<SUP> </SUP>(Ad3, Ad7, and Ad21)<SUP> </SUP>have simultaneously reemerged against<SUP> </SUP>the background of Ad4<SUP> </SUP>(figure 2). For the first<SUP> </SUP>time, an AdB2 serotype<SUP> </SUP>(Ad14) has emerged as<SUP> </SUP>a significant contributor to<SUP> </SUP>ARD in the Western<SUP> </SUP>Hemisphere. The simultaneous emergence<SUP> </SUP>of many serotypes has<SUP> </SUP>been broadly spread across<SUP> </SUP>many geographically isolated facilities,<SUP> </SUP>although Ad4 remains common<SUP> </SUP>at all sites. Population-based<SUP> </SUP>ARD rate data do<SUP> </SUP>not suggest that the<SUP> </SUP>emergence and subsequent dominance<SUP> </SUP>of Ad14 at San<SUP> </SUP>Diego in early 2006<SUP> </SUP>had any effect on<SUP> </SUP>the overall rate of<SUP> </SUP>ARD (figure 3).<SUP> </SUP>
Soon after the<SUP> </SUP>original identification of Ads<SUP> </SUP>as agents of adult<SUP> </SUP>ARD in the 1950s<SUP> </SUP>[16], 4 scattered ARD<SUP> </SUP>outbreaks associated with the<SUP> </SUP>AdB2 serotype 14 were<SUP> </SUP>reported in military and<SUP> </SUP>civilian adult populations. Ad14<SUP> </SUP>was initially discovered in<SUP> </SUP>an outbreak of ARD<SUP> </SUP>in Dutch recruits in<SUP> </SUP>1955 [21]. Civilian outbreaks<SUP> </SUP>were reported in Great<SUP> </SUP>Britain in 1955 [26],<SUP> </SUP>in Uzbekistan in 1962<SUP> </SUP>[27], and in Czechoslovakia<SUP> </SUP>in 1963 (the only<SUP> </SUP>sustained outbreak of Ad14<SUP> </SUP>ever previously recorded) [28].<SUP> </SUP>An apparent Ad11/Ad14 intermediate<SUP> </SUP>strain (denominated Ad14?11) was<SUP> </SUP>isolated after a large<SUP> </SUP>outbreak during a military<SUP> </SUP>training exercise in Spain<SUP> </SUP>in 1969 [29]. In<SUP> </SUP>general, AdB2 serotypes are<SUP> </SUP>more commonly associated with<SUP> </SUP>sporadic illness, most often<SUP> </SUP>kidney and urinary tract<SUP> </SUP>infections [3, 6].<SUP> </SUP>
Although both<SUP> </SUP>military and civilian surveys<SUP> </SUP>of Ad-associated ARD have<SUP> </SUP>commonly included assays capable<SUP> </SUP>of detecting and identifying<SUP> </SUP>Ad14 [30, 31], this<SUP> </SUP>serotype was not reported<SUP> </SUP>in large outbreaks of<SUP> </SUP>ARD between the 1960s<SUP> </SUP>and the turn of<SUP> </SUP>the century. Such surveys<SUP> </SUP>reported, for example, no<SUP> </SUP>Ad14 at all (0<SUP> </SUP>of 1792 Ad isolates<SUP> </SUP>from children with respiratory<SUP> </SUP>illness in the United<SUP> </SUP>States) [30] or very<SUP> </SUP>little (<1% of AdB<SUP> </SUP>serotypes other than Ad3<SUP> </SUP>or Ad7 among 3313<SUP> </SUP>Ad isolates from children<SUP> </SUP>and adults with respiratory<SUP> </SUP>illness in the United<SUP> </SUP>Kingdom) [31].<SUP> </SUP>
A few recent<SUP> </SUP>studies yielded greater numbers<SUP> </SUP>of AdB2 identifications. A<SUP> </SUP>survey of adults and<SUP> </SUP>children with ARD in<SUP> </SUP>Egypt yielded a surprising<SUP> </SUP>proportion of Ad11 (4%<SUP> </SUP>of isolated Ads) [6],<SUP> </SUP>and recent studies in<SUP> </SUP>Taiwan yielded similar proportions<SUP> </SUP>of Ad14 (2%?11% of<SUP> </SUP>isolates) [32, 33]. Ad11<SUP> </SUP>was recently identified in<SUP> </SUP>2 German military troops<SUP> </SUP>returning from an exercise<SUP> </SUP>in Turkey [22]. The<SUP> </SUP>affected troops had severe<SUP> </SUP>ARD symptoms and were<SUP> </SUP>strictly quarantined while the<SUP> </SUP>agent was identified. The<SUP> </SUP>exercise itself was subject<SUP> </SUP>to a widespread outbreak<SUP> </SUP>of ARD, which may<SUP> </SUP>well have been associated<SUP> </SUP>with the identified strain<SUP> </SUP>of Ad11.<SUP> </SUP>
These data point<SUP> </SUP>to a recent emergence<SUP> </SUP>of AdB2 serotypes as<SUP> </SUP>agents of ARD in<SUP> </SUP>healthy adults and children<SUP> </SUP>in Eurasia. This is<SUP> </SUP>similar to the current<SUP> </SUP>emergence of AdB2 in<SUP> </SUP>US military recruits, as<SUP> </SUP>described here. However, given<SUP> </SUP>that the serotypes involved<SUP> </SUP>are diverse, it seems<SUP> </SUP>unlikely that the simultaneous<SUP> </SUP>emergence of AdB2 serotypes<SUP> </SUP>as respiratory pathogens in<SUP> </SUP>multiple continents is resulting<SUP> </SUP>from the spread of<SUP> </SUP>recently evolved and adaptively<SUP> </SUP>superior strains.<SUP> </SUP>
The temporal distribution<SUP> </SUP>of Ads causing ARD<SUP> </SUP>in recruits can be<SUP> </SUP>highly varied. Sometimes single<SUP> </SUP>serotypes will dominate in<SUP> </SUP>a specific region, being<SUP> </SUP>very sharply replaced by<SUP> </SUP>other serotypes at intervals<SUP> </SUP>of a few years<SUP> </SUP>(see van der Veen<SUP> </SUP>et al. [34], for<SUP> </SUP>example). Sometimes 2 or<SUP> </SUP>3 types will share<SUP> </SUP>dominance in specific outbreaks;<SUP> </SUP>this is commonly seen<SUP> </SUP>with Ad3 and Ad7<SUP> </SUP>(see Ryan et al.<SUP> </SUP>[15], for example). Before<SUP> </SUP>the institution of vaccination<SUP> </SUP>protocols, subspecies B1 (Ad3<SUP> </SUP>and/or Ad7) and species<SUP> </SUP>E (Ad4) were often<SUP> </SUP>seen in significant numbers<SUP> </SUP>in the same year<SUP> </SUP>at the same site<SUP> </SUP>in US recruit facilities<SUP> </SUP>[35], and many vaccinated<SUP> </SUP>recruits with Ad-associated ARD<SUP> </SUP>were found to harbor<SUP> </SUP>species E and B<SUP> </SUP>serotypes simultaneously [36]. However,<SUP> </SUP>since the gradual withdrawal<SUP> </SUP>of vaccination measures in<SUP> </SUP>the United States between<SUP> </SUP>1996 and 1999, Ad4<SUP> </SUP>has dominated recruit training<SUP> </SUP>facilities. Ad4 has been<SUP> </SUP>associated with >98% of<SUP> </SUP>Ad-associated ARD cases from<SUP> </SUP>1999 through 2005 ([20]<SUP> </SUP>and figure 2), and AdE/AdB<SUP> </SUP>coinfections have been rare<SUP> </SUP>([36] and figure 2). The<SUP> </SUP>data presented here, which<SUP> </SUP>show many AdE/AdB coinfections<SUP> </SUP>associated with the general<SUP> </SUP>AdB reemergence in 2006,<SUP> </SUP>suggest that coinfections are<SUP> </SUP>the result of cocirculation<SUP> </SUP>and that neither coinfection<SUP> </SUP>nor cocirculation is directly<SUP> </SUP>related to vaccination status.<SUP> </SUP>
Coinfections<SUP> </SUP>are shown as split<SUP> </SUP>boxes in figure 2. Coinfections<SUP> </SUP>are important because they<SUP> </SUP>provide the opportunity for<SUP> </SUP>Ad strains to recombine<SUP> </SUP>and form new variants<SUP> </SUP>[6, 36]. Strains responsible<SUP> </SUP>for recent outbreak waves,<SUP> </SUP>including Ad7h and the<SUP> </SUP>genome type of Ad4<SUP> </SUP>that has dominated most<SUP> </SUP>of the recruit training<SUP> </SUP>facilities for the last<SUP> </SUP>10 years (Ad4a) [24],<SUP> </SUP>appear to be interserotypic<SUP> </SUP>recombinants [37, 38]. Coinfections<SUP> </SUP>with multiple Ads have<SUP> </SUP>only recently been recognized,<SUP> </SUP>owing to the advent<SUP> </SUP>of PCR typing [36].<SUP> </SUP>Immunological methods such as<SUP> </SUP>neutralization are often done<SUP> </SUP>with low challenge doses<SUP> </SUP>(100 TCID<SUB>50</SUB>) using single<SUP> </SUP>antisera and, as such,<SUP> </SUP>are unlikely to identify<SUP> </SUP>mixtures of multiple serotypes.<SUP> </SUP>
Ad4<SUP> </SUP>is ubiquitous in the<SUP> </SUP>recruits' physical environment, and<SUP> </SUP>transmission appears to be<SUP> </SUP>independent of regular import<SUP> </SUP>from outside sources to<SUP> </SUP>training centers [17]. During<SUP> </SUP>the past 10 years<SUP> </SUP>of high Ad4 prevalence,<SUP> </SUP>different genome types have<SUP> </SUP>predominated at different sites,<SUP> </SUP>with a pattern of<SUP> </SUP>circulation that suggests that<SUP> </SUP>Ad4 population dynamics are<SUP> </SUP>driven by internal, possibly<SUP> </SUP>environmental, reservoirs [24]. The<SUP> </SUP>data presented here suggest<SUP> </SUP>different dynamics for AdB<SUP> </SUP>serotypes. The emergence of<SUP> </SUP>Ad14, Ad21, and Ad7<SUP> </SUP>at multiple sites, all<SUP> </SUP>in the course of<SUP> </SUP>1 year and following<SUP> </SUP>a decade during which<SUP> </SUP>these types had little<SUP> </SUP>or no impact at<SUP> </SUP>all, suggests that these<SUP> </SUP>emergences are driven by<SUP> </SUP>importation from shared outside<SUP> </SUP>reservoirs. This reservoir is<SUP> </SUP>most likely the US<SUP> </SUP>civilian population, from which<SUP> </SUP>the affected trainees are<SUP> </SUP>recruited.<SUP> </SUP>
Dozens of publications from<SUP> </SUP>around the world during<SUP> </SUP>the last 40 years<SUP> </SUP>have documented the consistent<SUP> </SUP>circulation and clinical importance<SUP> </SUP>of Ad3, Ad7, and<SUP> </SUP>Ad21 in civilian populations.<SUP> </SUP>US recruit camps process<SUP> </SUP>tens of thousands of<SUP> </SUP>recruits from all parts<SUP> </SUP>of the United States<SUP> </SUP>every year. It would<SUP> </SUP>seem reasonable to assume<SUP> </SUP>that the influx of<SUP> </SUP>so many people from<SUP> </SUP>so many places would<SUP> </SUP>generate a constant influx<SUP> </SUP>of these common strains<SUP> </SUP>to the camps. It<SUP> </SUP>is therefore a mystery<SUP> </SUP>why all of the<SUP> </SUP>AdB1 serotypes that have<SUP> </SUP>ever been implicated in<SUP> </SUP>major ARD outbreaks in<SUP> </SUP>US recruits suddenly reemerged<SUP> </SUP>during the same year<SUP> </SUP>along with a previously<SUP> </SUP>rare AdB2 serotype.<SUP> </SUP>
We hypothesize<SUP> </SUP>that the main factor<SUP> </SUP>determining the variable presence<SUP> </SUP>of AdB serotypes among<SUP> </SUP>recruits is preexisting immunity<SUP> </SUP>in the recruit source<SUP> </SUP>population. In 1993, seropositivity<SUP> </SUP>for Ad7 among incoming<SUP> </SUP>recruits was estimated at<SUP> </SUP>27% [39]. A study<SUP> </SUP>from the postvaccination era,<SUP> </SUP>when Ad4 was dominant,<SUP> </SUP>estimated incoming recruit seropositivity<SUP> </SUP>for Ad7 at 61%<SUP> </SUP>[17]. The same 2<SUP> </SUP>studies reported a relatively<SUP> </SUP>constant level of incoming<SUP> </SUP>seropositivity for Ad4 (29%<SUP> </SUP>and 34%, respectively). Unfortunately,<SUP> </SUP>comparable data from 2006<SUP> </SUP>or from prevaccination periods<SUP> </SUP>of greater Ad diversity<SUP> </SUP>are not available. We<SUP> </SUP>hope that future work<SUP> </SUP>can address seropositivity on<SUP> </SUP>a more regular basis,<SUP> </SUP>because changes in source-population<SUP> </SUP>susceptibility may well predict<SUP> </SUP>the relative activity of<SUP> </SUP>different Ad serotypes in<SUP> </SUP>recruit facilities.<SUP> </SUP>
The previously deployed<SUP> </SUP>Ad4 and Ad7 vaccines,<SUP> </SUP>essentially identical to those<SUP> </SUP>currently in testing for<SUP> </SUP>future redeployment, do not<SUP> </SUP>directly target some of<SUP> </SUP>the currently circulating Ad<SUP> </SUP>serotypes. During previous periods<SUP> </SUP>of vaccination, Ad3 and<SUP> </SUP>Ad21 did continue to<SUP> </SUP>circulate among recruits, yet<SUP> </SUP>they never caused a<SUP> </SUP>return to prevaccination levels<SUP> </SUP>of disease. In fact,<SUP> </SUP>the vaccines appeared to<SUP> </SUP>prevent >95% of Ad-associated<SUP> </SUP>ARD and >50% of<SUP> </SUP>total recruit ARD despite<SUP> </SUP>the presence of these<SUP> </SUP>heterotypic Ads [18]. In<SUP> </SUP>studies using serum obtained<SUP> </SUP>from military recruits vaccinated<SUP> </SUP>with the Ad4 and<SUP> </SUP>Ad7 vaccines, Ad7 immunization<SUP> </SUP>was shown to generate<SUP> </SUP>a significant increase in<SUP> </SUP>levels of neutralizing antibodies<SUP> </SUP>against Ad3 and Ad14<SUP> </SUP>[40]. The cross-protective interaction<SUP> </SUP>between Ad7 and Ad14<SUP> </SUP>may be reflected in<SUP> </SUP>the data presented here<SUP> </SUP>(figure 2): Ad7 and Ad14<SUP> </SUP>are almost exclusively clustered<SUP> </SUP>at different sites. It<SUP> </SUP>will be important to<SUP> </SUP>track the response of<SUP> </SUP>different serotypes if and<SUP> </SUP>when the vaccines are<SUP> </SUP>redeployed. It would certainly<SUP> </SUP>be possible to develop<SUP> </SUP>vaccines against other ARD-associated<SUP> </SUP>Ads in the advent<SUP> </SUP>that currently circulating strains<SUP> </SUP>of Ad3, Ad14, or<SUP> </SUP>Ad21 are found to<SUP> </SUP>cause high rates of<SUP> </SUP>disease after reinstitution of<SUP> </SUP>the Ad4 and Ad7<SUP> </SUP>vaccines, although previous evidence<SUP> </SUP>suggested that the addition<SUP> </SUP>of Ad21 to the<SUP> </SUP>Ad4/Ad7 combination decreased the<SUP> </SUP>effectiveness of all 3<SUP> </SUP>[41].<SUP> </SUP>
Acknowledgments<SUP> </SUP>
We thank Dr. Leonard<SUP> </SUP>Binn for suggesting critical<SUP> </SUP>historical references. We acknowledge<SUP> </SUP>the administrative support of<SUP> </SUP>the Henry M. Jackson<SUP> </SUP>Foundation for Military Medicine<SUP> </SUP>and the efforts of<SUP> </SUP>the entire Naval Health<SUP> </SUP>Research Center team, especially<SUP> </SUP>the technicians and collection<SUP> </SUP>personnel whose efforts are<SUP> </SUP>represented in this work.<SUP> </SUP>We also acknowledge the<SUP> </SUP>clinic commanders and medical<SUP> </SUP>staff at Fort Benning,<SUP> </SUP>GA; Fort Jackson, SC;<SUP> </SUP>Fort Leonard Wood, MO<SUP> </SUP>(US Army); Lackland Air<SUP> </SUP>Force Base, San Antonio,<SUP> </SUP>TX (US Air Force);<SUP> </SUP>Naval Training Center, Great<SUP> </SUP>Lakes, IL (US Navy);<SUP> </SUP>Marine Corps Recruit Depot,<SUP> </SUP>San Diego, CA; Marine<SUP> </SUP>Corps Recruit Depot, Parris<SUP> </SUP>Island, SC (US Marine<SUP> </SUP>Corps); and Coast Guard<SUP> </SUP>Training Center, Cape May,<SUP> </SUP>NJ (US Coast Guard)<SUP> </SUP>for the permissions, access,<SUP> </SUP>and assistance necessary to<SUP> </SUP>conduct these studies.<SUP> </SUP>
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First citation in article | PubMed<SUP> </SUP></TD></TR>

</TABLE>

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

The Journal of Infectious Diseases 2007;196:1436-1437
This article is in the public domain, and no copyright is claimed.
0022-1899/2007/19610-0003$15.00
<HR><TABLE cellPadding=5><TBODY><TR><TD align=middle bgColor=#990000>EDITORIAL<SUP> </SUP>COMMENTARY</TD><SUP></SUP></TR></TBODY></TABLE>

Emergence<SUP> </SUP>of<SUP> </SUP>Adenovirus<SUP> </SUP>Type<SUP> </SUP>14<SUP> </SUP>in<SUP> </SUP>US<SUP> </SUP>Military<SUP> </SUP>Recruits?A<SUP> </SUP>New<SUP> </SUP>Challenge<SUP></SUP><SUP></SUP><SUP> </SUP>

Leonard N. Binn,<SUP>1</SUP><SUP> </SUP>Jose L. Sanchez,<SUP>2</SUP><SUP> </SUP>and<SUP> </SUP>Joel C. Gaydos<SUP>2</SUP><SUP> </SUP><SUP>1</SUP>Division<SUP> </SUP>of<SUP> </SUP>Viral<SUP> </SUP>Diseases,<SUP> </SUP>Walter<SUP> </SUP>Reed<SUP> </SUP>Army<SUP> </SUP>Institute<SUP> </SUP>of<SUP> </SUP>Research,<SUP> </SUP>and<SUP> </SUP><SUP>2</SUP>Department<SUP> </SUP>of<SUP> </SUP>Defense<SUP> </SUP>Global<SUP> </SUP>Emerging<SUP> </SUP>Infections<SUP> </SUP>Surveillance<SUP> </SUP>and<SUP> </SUP>Response<SUP> </SUP>System,<SUP> </SUP>Silver<SUP> </SUP>Spring,<SUP> </SUP>Maryland<SUP> </SUP>

</B>

Received 4 June 2007; accepted 4 June 2007; electronically published 31 October 2007.<SUP> </SUP>
Potential conflicts of interest:<SUP> </SUP>none reported.<SUP> </SUP>
The opinions or<SUP> </SUP>assertions contained herein are<SUP> </SUP>the private views of<SUP> </SUP>the authors and are<SUP> </SUP>not to be construed<SUP> </SUP>as official or as<SUP> </SUP>reflecting the true views<SUP> </SUP>of the Department of<SUP> </SUP>the Army or the<SUP> </SUP>Department of Defense.<SUP> </SUP>
(See the<SUP> </SUP>article by Metzgar et al., on<SUP> </SUP>pages 1465?73.)<SUP> </SUP>
Reprints<SUP> </SUP>or<SUP> </SUP>correspondence:<SUP> </SUP>Dr.<SUP> </SUP>Leonard<SUP> </SUP>N.<SUP> </SUP>Binn,<SUP> </SUP>Div.<SUP> </SUP>of<SUP> </SUP>Viral<SUP> </SUP>Diseases,<SUP> </SUP>Walter<SUP> </SUP>Reed<SUP> </SUP>Army<SUP> </SUP>Institute<SUP> </SUP>of<SUP> </SUP>Research,<SUP> </SUP>503<SUP> </SUP>Robert<SUP> </SUP>Grant<SUP> </SUP>Ave.,<SUP> </SUP>Silver<SUP> </SUP>Spring,<SUP> </SUP>MD<SUP> </SUP>20910<SUP> </SUP>(leonard.binn@na.amedd.army.mil).


<HR> In this issue<SUP> </SUP>of the <!--Start Italic-->Journal,<!--End Italic--> Metzgar<SUP> </SUP>et al. [1] report<SUP> </SUP>a significant addition to<SUP> </SUP>the adenovirus (Ad) types<SUP> </SUP>infecting US military recruits<SUP> </SUP>with acute respiratory disease<SUP> </SUP>(ARD). Their ongoing epidemiological<SUP> </SUP>studies, which use rapid<SUP> </SUP>molecular diagnostic procedures, enabled<SUP> </SUP>the detection and identification<SUP> </SUP>of a species B2<SUP> </SUP>Ad, type 14 (Ad14),<SUP> </SUP>that had not been<SUP> </SUP>previously observed in US<SUP> </SUP>military recruits. This virus<SUP> </SUP>was originally recovered >50<SUP> </SUP>years ago from Dutch<SUP> </SUP>recruits [2], with only<SUP> </SUP>2 subsequent reports of<SUP> </SUP>ARD associated with Ad14<SUP> </SUP>[1, 3]. In contrast<SUP> </SUP>to the B1 Ads<SUP> </SUP>(i.e., types 3, 7,<SUP> </SUP>and 21) that often<SUP> </SUP>infect the respiratory tract,<SUP> </SUP>Ad14 respiratory infections have<SUP> </SUP>been very rarely observed<SUP> </SUP>in adult military and<SUP> </SUP>civilian populations [4].<SUP> </SUP>
At present,<SUP> </SUP>studies are under way<SUP> </SUP>at military training centers<SUP> </SUP>to field test replacements<SUP> </SUP>for the live oral<SUP> </SUP>Ad4 and Ad7 vaccines<SUP> </SUP>that had been used<SUP> </SUP>for >25 years but<SUP> </SUP>were lost when production<SUP> </SUP>ceased in the 1990s<SUP> </SUP>[5?7]. The recent recovery<SUP> </SUP>of Ad14 raises new<SUP> </SUP>questions as to whether<SUP> </SUP>Ad14 will take hold<SUP> </SUP>as a respiratory disease<SUP> </SUP>agent in civilian and<SUP> </SUP>military populations and whether<SUP> </SUP>the candidate Ad4 and<SUP> </SUP>Ad7 vaccines will protect<SUP> </SUP>against Ad14. The absence<SUP> </SUP>of identified Ad14 respiratory<SUP> </SUP>infections in the United<SUP> </SUP>States suggests that we<SUP> </SUP>have highly susceptible civilian<SUP> </SUP>and military populations.<SUP> </SUP>
Review of<SUP> </SUP>past experience with the<SUP> </SUP>live oral Ad vaccines<SUP> </SUP>may be of value<SUP> </SUP>in assessing the long-term<SUP> </SUP>threat of Ad14 to<SUP> </SUP>recruits. Previous observation of<SUP> </SUP>responses to the Ad4<SUP> </SUP>and Ad7 vaccines indicated<SUP> </SUP>that they were highly<SUP> </SUP>effective against homologous viruses<SUP> </SUP>and Ad3 [6, 7].<SUP> </SUP>Vaccines responses to Ad3<SUP> </SUP>were attributed to some<SUP> </SUP>level of Ad3 occurring<SUP> </SUP>naturally and to Ad3<SUP> </SUP>being antigenically related to<SUP> </SUP>Ad7, both being species<SUP> </SUP>B1 viruses. Therefore, after<SUP> </SUP>exposure to the Ad7<SUP> </SUP>vaccine virus, heterotypic responses<SUP> </SUP>to Ad3 occurred [8].<SUP> </SUP>In contrast to Ad3,<SUP> </SUP>naturally occurring background infections<SUP> </SUP>with Ad21, also a<SUP> </SUP>species B1 virus, have<SUP> </SUP>been considered rare. Recent<SUP> </SUP>studies of recruits with<SUP> </SUP>ARD found only small<SUP> </SUP>numbers infected with Ad21<SUP> </SUP>[1]. Only a few<SUP> </SUP>additional Ad21 infections were<SUP> </SUP>identified via antibody testing,<SUP> </SUP>suggesting that the virus<SUP> </SUP>currently is not highly<SUP> </SUP>communicable [9, 10]. However,<SUP> </SUP>in 1975?1976, Ad21 was<SUP> </SUP>recovered from large numbers<SUP> </SUP>of recruits with ARD<SUP> </SUP>who had been vaccinated<SUP> </SUP>against Ad4 and Ad7.<SUP> </SUP>ARD-associated Ad21 infections continued<SUP> </SUP>for months at most<SUP> </SUP>training facilities, resulting in<SUP> </SUP>the testing of an<SUP> </SUP>experimental live oral vaccine<SUP> </SUP>[11, 12]. However, long-term<SUP> </SUP>observation revealed that Ad21<SUP> </SUP>did not persist as<SUP> </SUP>a significant cause of<SUP> </SUP>ARD, even though outbreaks<SUP> </SUP>occurred in the 1970s<SUP> </SUP>and again in 1985<SUP> </SUP>[13]. Thus, there has<SUP> </SUP>been no interest in<SUP> </SUP>renewing studies of an<SUP> </SUP>Ad21 vaccine.<SUP> </SUP>
It is possible<SUP> </SUP>that Ad14 and other<SUP> </SUP>Ads could cause significant<SUP> </SUP>ARD morbidity among military<SUP> </SUP>recruits for a limited<SUP> </SUP>period and then disappear<SUP> </SUP>as an important cause<SUP> </SUP>of respiratory disease. It<SUP> </SUP>is also possible that<SUP> </SUP>some protection against emerging<SUP> </SUP>Ads could be provided<SUP> </SUP>as a result of<SUP> </SUP>heterotypic antibody responses to<SUP> </SUP>other viruses, including the<SUP> </SUP>Ad7 vaccine virus. Past<SUP> </SUP>studies in The Netherlands<SUP> </SUP>found that Ad14-associated ARD<SUP> </SUP>did not persist in<SUP> </SUP>their recruits [2, 14].<SUP> </SUP>Additionally, the development of<SUP> </SUP>heterotypic antibody to Ad14<SUP> </SUP>after Ad7 immunization was<SUP> </SUP>reported in 1960 [15].<SUP> </SUP>
Contemporary<SUP> </SUP>serologic surveys of new<SUP> </SUP>US military recruits will<SUP> </SUP>provide important information on<SUP> </SUP>their susceptibility. Testing archival<SUP> </SUP>serum-bank specimens from recruits<SUP> </SUP>vaccinated against Ad4 and<SUP> </SUP>Ad7 and from recruits<SUP> </SUP>with ARD who had<SUP> </SUP>laboratory confirmation of a<SUP> </SUP>specific Ad type could<SUP> </SUP>provide data on the<SUP> </SUP>occurrence of heterotypic antibody<SUP> </SUP>responses to Ad14. Additionally,<SUP> </SUP>measurement of Ad14-associated ARD<SUP> </SUP>during the current Ad4<SUP> </SUP>and Ad7 vaccine trials<SUP> </SUP>will be important, as<SUP> </SUP>will measurement of immunity<SUP> </SUP>to Ad14 associated with<SUP> </SUP>immunization. Molecular studies should<SUP> </SUP>be conducted to characterize<SUP> </SUP>recovered Ad14 isolates and<SUP> </SUP>to determine whether changes<SUP> </SUP>may have occurred in<SUP> </SUP>the genome that could<SUP> </SUP>affect communicability and pathogenicity,<SUP> </SUP>a situation that was<SUP> </SUP>described in Spanish recruits<SUP> </SUP>in 1976 [16].<SUP> </SUP>
The events<SUP> </SUP>described by Metzgar et<SUP> </SUP>al. provide strong support<SUP> </SUP>for ongoing, laboratory-based surveillance<SUP> </SUP>in military training populations.<SUP> </SUP>Because little is known<SUP> </SUP>about the dynamics involved<SUP> </SUP>in the emergence of<SUP> </SUP>important acute febrile respiratory<SUP> </SUP>disease pathogens in military<SUP> </SUP>populations, studies describing agent<SUP> </SUP>persistence and transmission within<SUP> </SUP>military communities and surveillance<SUP> </SUP>of associated civilian communities<SUP> </SUP>should be continued. Whether<SUP> </SUP>Ad14 will persist as<SUP> </SUP>a significant cause of<SUP> </SUP>ARD in recruits cannot<SUP> </SUP>be predicted. Therefore, it<SUP> </SUP>is advisable to preserve<SUP> </SUP>candidate specimens of Ad14<SUP> </SUP>for passage in suitable<SUP> </SUP>cells for vaccine seed<SUP> </SUP>virus and molecular studies.<SUP> </SUP>
After<SUP> </SUP>the licensure of the<SUP> </SUP>Ad4 and Ad7 vaccines<SUP> </SUP>currently being tested, complacency<SUP> </SUP>regarding the control of<SUP> </SUP>acute febrile respiratory diseases<SUP> </SUP>in military populations cannot<SUP> </SUP>occur. Continued surveillance will<SUP> </SUP>be necessary to assess<SUP> </SUP>the effectiveness of the<SUP> </SUP>new vaccines and to<SUP> </SUP>detect the presence of<SUP> </SUP>emergent Ads, influenza viruses,<SUP> </SUP>and other respiratory agents<SUP> </SUP>that cause morbidity and<SUP> </SUP>interfere with military training.<SUP> </SUP>The application of the<SUP> </SUP>modern tools of molecular<SUP> </SUP>biology should greatly enhance<SUP> </SUP>our understanding of these<SUP> </SUP>agents and the dynamics<SUP> </SUP>of acute febrile respiratory<SUP> </SUP>diseases in the unique<SUP> </SUP>environment of military recruits<SUP> </SUP>undergoing their initial entry<SUP> </SUP>(i.e., basic) military training.<SUP> </SUP>
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First citation in article<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>7. </TD><TD vAlign=top align=left>Gaydos CA, Gaydos JC. Adenovirus vaccine. In: Plotkin SA, Orenstein WA, eds. Vaccines. 4th ed. Philadelphia: Saunders, 2004:863?85. First citation in article<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>8. </TD><TD vAlign=top align=left>Grayston T, Loosli CG, Johnston PB, Smith ME, Woolridge RL. Neutralization and complement fixing antibody response to adenovirus infection. J Infect Dis 1956; 99:199?206. First citation in article | PubMed<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>9. </TD><TD vAlign=top align=left>Kolavic-Gray SA, Binn LN, Sanchez JL, et al. Large epidemic of adenovirus type 4 infection among military trainees: epidemiological, clinical, and laboratory studies. Clin Infect Dis 2002; 35:808?18. First citation in article | PubMed<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>10. </TD><TD vAlign=top align=left>Vento TJ, Houng H-S, Gaydos JC, Sun W, Kuschner R, Binn L. Adenovirus type 4 loads on throat and hand specimens from military basic trainees [abstract 343]. In: Program and abstracts of the 41st Annual Meeting of the Infectious Diseases Society of America (San Diego). Arlington, VA: Infectious Diseases Society of America, 2003. First citation in article<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>11. </TD><TD vAlign=top align=left>Takafuji ET, Gaydos JC, Allen RG, Top FH Jr. Simultaneous administration of live, enteric-coated adenovirus types 4, 7, and 21 vaccines: safety and immunogenicity. J Infect Dis 1979; 140:48?53. First citation in article | PubMed<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>12. </TD><TD vAlign=top align=left>Top FH Jr, Brandt WE, Russell PK. Adenovirus ARD in basic combat trainees. In: Research in biological and medical sciences: annual progress report, 1975/1976. Washington, DC: Walter Reed Army Institute of Research, 1976:462?5. First citation in article<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>13. </TD><TD vAlign=top align=left>Gaydos CA, Gaydos JC. Adenovirus vaccines in the US military. Mil Med 1995; 160:300?4. First citation in article | PubMed<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>14. </TD><TD vAlign=top align=left>van der Veen J, Oie KG, Abarbanel MFW. Patterns of infections with adenovirus types 4, 7 and 21 in military recruits during a 9-year survey. J Hyg (Lond) 1969; 67:255?68. First citation in article | PubMed<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>15. </TD><TD vAlign=top align=left>van der Veen J, Prins A. Studies on the significance of the recall phenomenon in the antibody response to adenovirus vaccine and infection. J Immunol 1960; 84:562?8. First citation in article | PubMed<SUP> </SUP></TD></TR><TR><TD vAlign=top align=right>16. </TD><TD vAlign=top align=left>Hierholzer JC, Pumarola A. Antigenic characterization of intermediate adenovirus 14-11 strains associated with upper respiratory illness in a military camp. Infect Immun 1976; 13:354?9. First citation in article | PubMed<SUP> </SUP></TD></TR>

</TABLE>

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

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gi|153861616|gb|EF503568|[153861616]
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[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

Interesting that out of 88 cases, 15 had coinfections!

and...



.

The sample to the<SUP> </SUP>right of the asterisk<SUP> </SUP>was a triple infection<SUP> </SUP>with Ad4, Ad7, and<SUP> </SUP>Ad14.

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

Interesting that out of 88 cases, 15 had coinfections!

and...



.

In March<SUP> </SUP>and April of 2006,<SUP> </SUP>Ad14 (subspecies B2) simultaneously<SUP> </SUP>emerged at 5 training<SUP> </SUP>centers, including San Diego,<SUP> </SUP>Lackland, Fort Leonard Wood,<SUP> </SUP>Great Lakes, and Fort<SUP> </SUP>Benning, thereby affecting all<SUP> </SUP>services except the Coast<SUP> </SUP>Guard (Cape May). At<SUP> </SUP>3 of these centers,<SUP> </SUP>Ad14 first appeared in<SUP> </SUP>coinfections with AdB1 serotypes<SUP> </SUP>and in 1 case<SUP> </SUP>as part of a<SUP> </SUP>triple infection with Ad4<SUP> </SUP>and Ad7. Ad14 maintained<SUP> </SUP>a consistent presence at<SUP> </SUP>Fort Benning, San Diego,<SUP> </SUP>and Great Lakes during<SUP> </SUP>summer 2006. At the<SUP> </SUP>same time, a variety<SUP> </SUP>of AdB1 serotypes emerged<SUP> </SUP>at all sites except<SUP> </SUP>Cape May. Ad3 cases<SUP> </SUP>were clustered at Fort<SUP> </SUP>Jackson, Ad7 at Fort<SUP> </SUP>Leonard Wood and Parris<SUP> </SUP>Island, and Ad21 at<SUP> </SUP>Parris Island and Fort<SUP> </SUP>Benning. All 6 identified<SUP> </SUP>serotypes were seen at<SUP> </SUP>Fort Benning during one<SUP> </SUP>2-month period. Many coinfections<SUP> </SUP>were identified, the majority<SUP> </SUP>of which were coinfections<SUP> </SUP>with Ad4 and one<SUP> </SUP>of the species B<SUP> </SUP>serotypes.<SUP> </SUP>

[HenryN]

Re: CDC: New Respiratory Bug Has Killed 10

Interesting that out of 88 cases, 15 had coinfections!

and...



.

Coinfections<SUP> </SUP>are important because they<SUP> </SUP>provide the opportunity for<SUP> </SUP>Ad strains to recombine<SUP> </SUP>and form new variants<SUP> </SUP>[6, 36]. Strains responsible<SUP> </SUP>for recent outbreak waves,<SUP> </SUP>including Ad7h and the<SUP> </SUP>genome type of Ad4<SUP> </SUP>that has dominated most<SUP> </SUP>of the recruit training<SUP> </SUP>facilities for the last<SUP> </SUP>10 years (Ad4a) [24],<SUP> </SUP>appear to be interserotypic<SUP> </SUP>recombinants [37, 38]. Coinfections<SUP> </SUP>with multiple Ads have<SUP> </SUP>only recently been recognized,<SUP> </SUP>owing to the advent<SUP> </SUP>of PCR typing [36].<SUP> </SUP>