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For communicable diseases, clinical management, and public health response, it is often important to know the body's immune response following exposure and infection with pathogens. Although humoral and cell-mediated immunity both play roles in the body's specific immunity against viral pathogens, testing antibody response for humoral immunity is much more common and is also easier in clinical virology laboratories than testing for cell-mediated immunity because of the convenience of antibody serological testing methods. There is a long history of using various serologic methods in clinical virology laboratories for antibody detection. Some methods, such as complement fixation test and immunodiffusion test, have been gradually phased out and replaced by faster and less laborious methods (1, 2). In this chapter, we will focus on antibody detection methods used in clinical virology laboratories: neutralization, hemagglutination inhibition, indirect immunofluorescence, enzyme immunoassay, and Western blot.

The development of a quantitative real-time PCR (qPCR) assay for human rhinovirus serotype 16 (HRV16) is described using the plasmid pR16.11, which contains the full-length genome of HRV16. A standard curve was generated by plotting the critical threshold (Ct) against numbers of plasmid. The limit of sensitivity was less than10 cDNA copies, and the curve showed a high degree of linearity over a range of 101 to 106 cDNA copies with r2 ≥ 0.9989. Amplification efficiency of the qPCR was greater than 97.6 percent. The standard curve was highly reproducible with low intra- and inter-assay coefficients of variation. Standard curves were also generated from cDNA derived from two viral suspensions of known TCID50, and were exactly parallel to those generated from the plasmid. Comparison of the curves generated from the plasmid or viral cDNA showed that for the two suspensions, TCID50 corresponded to either 142 or 2088 viral particles. This new qPCR will permit quantitative assessments of interactions between virus and epithelium such as determinations of the affinity and number of viral binding sites or of the number of virus produced per infected cell.

Background. First isolated in the Netherlands in 1955 during an outbreak of acute respiratory disease (ARD) among military recruits, human adenovirus 14 (HAdV-14) has historically been considered rare. With no precedent of circulation in North America, HAdV-14 has been isolated from military and civilian cases of ARD of variable severity since 2003 in the United States. Methods. Ninety-nine isolates from military and civilian cases from different geographic locations and circulation periods were characterized by restriction enzyme analysis of viral DNA and select gene sequencing. Results. All examined viruses were found to be identical and to belong to a new genome type designated "HAdV-14pl" (formerly known as "14a"). Comparative alignments of EIA, hexon, and fiber gene sequences with other subspecies B2 HAdVs suggest that HAdV-14p1, like the closely related HAdV-11a, arose from recombination among similar HAdV-11 and HAdV-14 ancestral strains. A deletion of 2 amino acids in the knob region of the fiber protein is the only identified unique characteristic of HAdV-14pl. Conclusion. The current geographic distribution of HAdV-14pl involves at least 15 states in the Unites States. The role of the fiber mutations in the recent emergence of HAdV-14pl ARD in North America warrants further study.

By using random PCR amplification, shotgun sequencing and sequence similarity searches, we analysed nucleic acids present in cell cultures inoculated with samples from unexplained cases of encephalitis. We identified a divergent human papillomavirus (HPV) sequence originating from a rectal swab. The full genome was amplified by inverse PCR and sequenced. The prototype of the sixth gammapapillomavirus species, HPV116, was not found in the patient's cerebrospinal fluid or respiratory secretions, nor in culture supernatants from other unexplained cases of encephalitis, indicating that its identification in an encephalitis patient was accidental.

Enteroviruses (EVs) are common seasonal viruses that are associated with a variety of diseases. High-quality monoclonal antibodies (MAbs) are needed to improve the accuracy of EV diagnosis in clinical laboratories. In the present study, the full-length VP1 genes of poliovirus 1 (Polio 1) and coxsackievirus B3 (Cox B3) were cloned, and the encoded proteins were expressed and used as antigens in an attempt to raise broad-spectrum MAbs to EVs. Two pan-EV MAbs were isolated: one raised against Polio 1 VP1 and the other against Cox B3 VP1. The binding sites of both pan-EV MAbs were mapped to an amino acid sequence within a conserved region in the N terminus of Polio 1 VP1 by peptide and competition enzyme-linked immunosorbent assay. Two additional MAbs, an EV70-specific MAb and an EV71/Cox A16-bispecific MAb, developed against EV70 and 71 VP1 proteins, were pooled with the two pan-EV MAbs (pan-EV MAb mix) and tested for their sensitivity and specificity in the staining of various virus-infected cells. The pan-EV MAb mix detected all 40 prototype EVs tested and showed no cross-reactivity to 18 different non-EV human viruses. Compared with two commercially available EV tests, the pan-EV MAb mix exhibited higher specificity than one test and broader spectrum reactivity than the other. Thus, our study demonstrates that full-length Polio 1 VP1 and Cox B3 VP1 can serve as effective antigens for developing a pan-EV MAb and that the pan-EV MAb mix can be used for the laboratory diagnosis of a wide range of EV infections.

Cardioviruses comprise a genus of picornaviruses that cause severe illnesses in rodents, but little is known about the prevalence, diversity, or spectrum of disease of such agents among humans. A single cardiovirus isolate, Saffold virus, was cultured in 1981 in stool from an infant with fever. Here, we describe the identification of a group of human cardioviruses that have been cloned directly from patient specimens, the first of which was detected using a pan-viral microarray in respiratory secretions from a child with influenza-like illness. Phylogenetic analysis of the nearly complete viral genome (7961 bp) revealed that this virus belongs to the Theiler's murine encephalomyelitis virus (TMEV) subgroup of cardioviruses and is most closely related to Saffold virus. Subsequent screening by RT-PCR of 719 additional respiratory specimens [637 (89%) from patients with acute respiratory illness] and 400 cerebrospinal fluid specimens from patients with neurological disease (aseptic meningitis, encephalitis, and multiple sclerosis) revealed no evidence of cardiovirus infection. However, screening of 751 stool specimens from 498 individuals in a gastroenteritis cohort resulted in the detection of 6 additional cardioviruses (1.2%). Although all 8 human cardioviruses (including Saffold virus) clustered together by phylogenetic analysis, significant sequence diversity was observed in the VP1 gene (66.9%–100% pairwise amino acid identities). These findings suggest that there exists a diverse group of novel human Theiler's murine encephalomyelitis virus-like cardioviruses that hitherto have gone largely undetected, are found primarily in the gastrointestinal tract, can be shed asymptomatically, and have potential links to enteric and extraintestinal disease.

More than 35 years ago, population-based studies of viral respiratory illnesses among US families were conducted in Ohio [1], Kansas [2], Louisiana [3], New York [4], and Washington [5]. As a result of these investigations, scientists concluded that adenovirus infection was quite common among children. Approximately 50% of infections were asymptomatic, and symptomatic infections were typically mild and resolved without sequelae. In contrast, military populations experienced severe epidemics of acute respiratory disease, including pneumonia and encephalitis, especially involving adenovirus types 4, 7, and 21. For example, in 1958, adenoviral infection was reported to have caused hospitalization of an estimated 10% of military recruits [6] and to be the etiology of most respiratory disease during winter months. Subsequently, vaccines for adenovirus types 4 and 7 were developed and effectively used among US military trainees from the 1970s until the late 1990s [7, 8]. Thus, until recently, adenovirus infection was considered to have little consequence, except for causing morbidity among military trainees. However, much has changed since these early epidemiological studies were conducted. In contrast to the modest number of adenovirus types recognized 35 years ago, 51 unique serotypes are now recognized. Different serotypes have been found to have different tissue tropisms that correlate with different clinical manifestations of infection. Limited epidemiological investigations have revealed that, among some specific serotypes, multiple genetic variants exist that often have quite different geographical distributions and associated virulence [9-11]. In addition, largely because of molecular diagnostics, adenovirus infection has been associated with a number of acute and chronic diseases, including chronic airway obstruction [12] and pulmonary dysplasia [13], myocarditis and dilated cardiomyopathy [14], mononucleosis-like syndromes [15], intussusception [16], sudden infant perinatal death [17], and obesity [18, 19]. Among some population groups, adenoviral infection is common and, often, severe. For example, the incidence of adenoviral disease among bone marrow transplant recipients varies from 3% to 20% [20], and mortality can exceed 50% [21]. Similarly, multiple recent outbreaks of adenoviral infection in the United States have frequently occurred among institutionalized children and in medical settings, resulting in significant morbidity and mortality among patients and medical staff [22]. In vitro studies suggest that specific adenovirus types are more likely to respond to certain antiviral therapies [23]. Finally, since the military lost its manufacturer of adenovirus types 4 and 7 vaccines in the late 1990s, numerous outbreaks of adenovirus infection have occurred among military trainees, resulting in great morbidity [24, 25]. Subsequently, the US Department of Defense identified a new vaccine manufacturer, and restoration of adenovirus types 4 and 7 vaccines is projected for 2008 [25]. It seems prudent to investigate whether previously very effective and safe vaccines may also benefit some nonmilitary populations. Therefore, an updated look at the epidemiology of human adenovirus infection is warranted. In this report, we present 25 months of viral and patient epidemiological data on clinical adenovirus infection detected through a nationwide network of 22 US military and civilian medical facilities. We used a recently described molecular adenovirus typing technique to characterize the strains of adenovirus [26].

Background Influenza surveillance is valuable for monitoring trends in influenza-related morbidity and mortality. Using the 2005–2006 influenza season as an example, this paper describes a comprehensive influenza surveillance program used by the California Department of Public Health (CDPH). Methods Data collected from patients evaluated for acute respiratory illness in a given week were reported and summarized the following week, including (1) electronic hospital pneumonia and influenza admission and antiviral usage records from Kaiser Permanente, (2) sentinel provider influenza-like illness (ILI) reports, (3) severe pediatric influenza case reports (e.g., children either hospitalized in intensive care or expired), (4) school clinic ILI evaluations, and (5) positive influenza test results from a network of academic, hospital, commercial, and public health laboratories and the state CDPH Viral and Rickettsial Disease Laboratory. Results Influenza activity in California in the 2005–2006 season was moderate in severity; all clinical and laboratory markers rose and fell consistently. Extensive laboratory characterization identified the predominant circulating virus strain as A/California/7/2004(H3N2), which was a component of the 2005–2006 influenza vaccine; 96% of samples tested showed adamantane resistance. Conclusions By using multiple, complementary surveillance methods coupled with a strong laboratory component, the CDPH has developed a simple, flexible, stable, and widely accepted influenza surveillance system that can monitor trends in statewide influenza activity, ascertain the correlation between circulating strains with vaccine strains, and assist with detection of new strain variants. The methods described can serve as a model for influenza surveillance in other states.

Human metapneumovirus (hMPV), a recently discovered paramyxovirus, is thought to be primarily a winter-spring pathogen affecting young children with a clinical presentation similar to that of respiratory syncytial virus. In June-July 2006, a respiratory outbreak in a long-term-care facility was reported to the local health department and investigated. Surveillance identified 26 residents and 13 staff with acute respiratory illness; 8 residents (31%) developed radiographically confirmed pneumonia, and 2 (5%) were hospitalized. Five of 14 respiratory specimens were positive by polymerase chain reaction assay for hMPV; sequencing identified genotype A. In institutionalized elderly persons, hMPV may be an important cause of respiratory outbreaks year-round.

An unidentified agent was cultured in primary monkey cells at the Los Angeles County Public Health Department from each of five stool specimens submitted from an outbreak of gastroenteritis. Electron microscopy and an adenovirus-specific monoclonal antibody confirmed this agent to be an adenovirus. Since viral titers were too low, complete serotyping was not possible. Using the DNase-sequence-independent viral nucleic acid amplification method, we identified several nucleotide sequences with a high homology to human adenovirus 41 (HAdV-41) and simian adenovirus 1 (SAdV-1). However, using anti-SAdV-1 sera, it was determined that this virus was serologically different than SAdV-1. Genomic sequencing and phylogenetic analysis confirmed that this new adenovirus was so divergent from the known human adenoviruses that it was not only a new type but also represented a new species (human adenovirus G). In a retrospective clinical study, this new virus was detected by PCR in one additional patient from a separate gastroenteritis outbreak. This study suggests that HAdV-52 may be one of many agents causing gastroenteritis of unknown etiology.

Background Human rhinoviruses (HRVs) are the most frequent cause of acute upper respiratory tract infection, however, they are also known to replicate in the lower respiratory tract and associate with more severe respiratory illnesses. An outbreak of HRV occurred in a long-term facility in Santa Cruz, California with unusually high morbidity and mortality. Objectives To identify viral characteristics associated with this unique outbreak, genetic relationships between these clinical isolates (SCRVs) and prototype strains of rhinovirus were investigated. Study design Sequence homology and phylogenetic analyses of the SCRV VP4/VP2 region were performed in conjunction with all HRV prototypes. Due to the importance of the 5′noncoding region (NCR) and the structural genes to viral replication and host immune responses, respectively, we focused on a segment of the HRV genome which includes these regions. Molecular models of SCRV were also assessed. Results SCRV showed closest similarity to HRV82 with some divergence from the prototype. Amino acid differences were concentrated within predicted neutralization epitopes within VP2, VP3 and VP1. Conclusion Sequence analyses and differences in cell culture growth characteristics suggest that this virus is a variant of HRV which has distinctive properties from its respective prototype strain.

Human parechoviruses (HPeV), members of the Parechovirus genus of Picornaviridae , are frequent pathogens but have been comparatively poorly studied, and little is known of their diversity, evolution, and molecular biology. To increase the amount of information available, we have analyzed 7 HPeV strains isolated in California between 1973 and 1992. We found that, on the basis of VP1 sequences, these fall into two genetic groups, one of which has not been previously observed, bringing the number of known groups to five. While these correlate partly with the three known serotypes, two members of the HPeV2 serotype belong to different genetic groups. In view of the growing importance of molecular techniques in diagnosis, we suggest that genotype is an important criterion for identifying viruses, and we propose that the genetic groups we have defined should be termed human parechovirus types 1 to 5. Complete nucleotide sequence analysis of two of the Californian isolates, representing two types, confirmed the identification of a new genetic group and suggested a role for recombination in parechovirus evolution. It also allowed the identification of a putative HPeV1 cis -acting replication element, which is located in the VP0 coding region, as well as the refinement of previously predicted 5′ and 3′ untranslated region structures. Thus, the results have significantly improved our understanding of these common pathogens.

Fever of unknown origin (FUO) is a serious problem in the United States. An unidentified agent was cultured from the stool of an infant who presented with FUO. This virus showed growth in HFDK cells and suckling mice. Using DNase sequence-independent single-primer amplification, we identified several nucleotide sequences with a high homology to Theiler's murine encephalomyelitis virus. Nearly full-length viral genome sequencing and phylogenetic analysis demonstrate that this virus is a member of the Cardiovirus genus of the Picornaviridae family.

Wild-type rubella viruses are genetically classified into 2 clades and 10 intraclade genotypes, of which 3 are provisional. The genotypes of 118 viruses from the United States were determined by sequencing part of the E1 coding region of these viruses and comparing the resulting sequences with reference sequences for each genotype, using the Bayesian inference program MRBAYES. Three genotypes of rubella viruses were found in the United States too infrequently to be considered for indigenous transmission. A fourth genotype was found frequently until 1981, and a fifth genotype was found frequently until 1988, but neither was obtained from nonimported cases after 1988. A sixth genotype was found frequently during 1996-2000, likely because of multiple importations from neighboring countries. The results of the present genetic analysis of rubella viruses found in the United States are consistent with elimination of indigenous viruses by 2001, the year when rubella was considered to be eliminated on the basis of epidemiological evidence.

Rhinovirus (RV) infection is the major cause of common colds and of asthma exacerbations. Because the epithelial cell layer is the primary target of RV infection, we hypothesize that RV-induced airway disease is associated with the perturbation of airway epithelial gene expression. In this study, well differentiated primary human airway epithelial cells were infected with either RV16 (major group) or RV1B (minor group). Transcriptional gene profiles from RV-infected and mock-infected control cells were analyzed by Affymetrix Genechip, and changes of the gene expression were confirmed by real-time RT-PCR analysis. At 24 h after infection, 48 genes induced by both viruses were identified. Most of these genes are related to the IFN pathway, and have been documented to have antiviral functions. Indeed, a significant stimulation of IFN-beta secretion was detected after RV16 infection. Neutralizing antibody specific to IFN-beta and a specific inhibitor of the Janus kinase pathway both significantly blocked the induction of RV-inducible genes. Further studies demonstrated that 2-aminopurine, a specific inhibitor double-stranded RNA-dependent protein kinase, could block both IFN-beta production and RV-induced gene expression. Thus, IFN-beta-dependent pathway is a part of the double-stranded RNA-initiated pathway that is responsible for RV-induced gene expression. Consistent with its indispensable role in the induction of antiviral genes, deactivation of this signaling pathway significantly enhanced viral production. Because increase of viral yield is associated with the severity of RV-induced airway illness, the discovery of an epithelial antiviral signaling pathway in this study will contribute to our understanding of the pathogenesis of RV-induced colds and asthma exacerbations.

Clinical similarities and shared seasonality suggested a relationship between adenovirus infection and Kawasaki disease. We performed adenovirus serology and quantitative polymerase chain reaction for both adenovirus and adeno-associated virus in patients with acute Kawasaki disease. No evidence was found to suggest a link between either virus and Kawasaki disease.

An outbreak of myocarditis occurred among adults in Illinois in 2003. Diagnostic testing of myocardial tissues from 3 patients and comprehensive tests for enterovirus and adenovirus of other specimens from patients were inconclusive. Appropriate specimen collection from patients with idiopathic cardiomyopathy and further enhancement of diagnostic techniques are needed.

During a 6-week period in 2003, 56 residents and 26 staff developed respiratory illness in a long-term facility; 12 residents died. Seven of 13 respiratory specimens were culture-positive for rhinovirus; 6 of the isolates were serotype 82. In elderly populations, severe illness may be associated with organisms typically considered to be “benign,” such as rhinovirus.

Enterovirus (EV) 68 was originally isolated in California in 1962 from four children with respiratory illness. Since that time, reports of EV68 isolation have been very uncommon. Between 1989 and 2003, 12 additional EV68 clinical isolates were identified and characterized, all of which were obtained from respiratory specimens of patients with respiratory tract illnesses. No EV68 isolates from enteric specimens have been identified from these same laboratories. These recent isolates, as well as the original California strains and human rhinovirus (HRV) 87 (recently shown to be an isolate of EV68 and distinct from the other human rhinoviruses), were compared by partial nucleotide sequencing in three genomic regions (partial sequencing of the 5'-non-translated region and 3D polymerase gene, and complete sequencing of the VP1 capsid gene). The EV68 isolates, including HRV87, were monophyletic in all three regions of the genome. EV68 isolates and HRV87 grew poorly at 37 degrees C relative to growth at 33 degrees C and their titres were reduced by incubation at pH 3.0, whereas the control enterovirus, echovirus 11, grew equally well at 33 and 37 degrees C and its titre was not affected by treatment at pH 3.0. Acid lability and a lower optimum growth temperature are characteristic features of the human rhinoviruses. It is concluded that EV68 is primarily an agent of respiratory disease and that it shares important biological and molecular properties with both the enteroviruses and the rhinoviruses.

In California, molecular testing was useful in decreasing suspicion for severe acute respiratory syndrome (SARS), by detecting common respiratory pathogens (influenza A/B, human metapneumovirus, picornavirus, Mycoplasma pneumoniae, Chlamydia spp., parainfluenza virus, respiratory syncytial virus, and adenovirus) in 23 (45%) of 51 patients with suspected SARS and 9 (47%) of 19 patients with probable SARS.

Genetic variation among 166 isolates of human adenovirus 7 (Ad7) obtained from 1966 to 2000 from the United States and Eastern Ontario, Canada, was determined by genome restriction analysis. Most (65%) isolates were identified as Ad7b. Two genome types previously undocumented in North America were also identified: Ad7d2 (28%), which first appeared in 1993 and was later identified throughout the Midwest and Northeast of the United States and in Canada; and Ad7h (2%), which was identified only in the U.S. Southwest in 1998 and 2000. Since 1996, Ad7d2 has been responsible for several civilian outbreaks of Ad7 disease and was the primary cause of a large outbreak of respiratory illness at a military recruit training center. The appearance of Ad7d2 and Ad7h in North America represents recent introduction of these viruses from previously geographically restricted areas and may herald a shift in predominant genome type circulating in the United States.

A single‐step multiplex reverse transcription‐polymerase chain reaction (RT‐PCR) for the simultaneous detection of influenza virus type and subtypes was evaluated for its use in clinical specimens. One part of each specimen was tested using an established standard culture and/or monoclonal antibody‐based immunofluorescence assay method, and the other part was tested by the multiplex RT‐PCR method for the presence or absence of the influenza virus, and its type and subtype. Sixty‐seven specimens were examined. The results revealed a strong agreement between the data obtained by the established method and those obtained by the multiplex RT‐PCR. J. Clin. Lab. Anal. 16:163–166, 2002. © 2002 Wiley‐Liss, Inc.

Enteroviruses (EV) have traditionally been identified by using serotype-specific antisera in a virus-neutralization test. Three EV strains isolated in California, USA, in 1955, 1964 and 1978, and a 1995 Oman isolate, were found to be antigenically related to one another; however, the strains were not neutralized by standard EV typing antisera, suggesting that they may represent a new EV serotype. The isolates were characterized genetically by RT–PCR coupled with amplicon sequencing and comparison to a database of enterovirus nucleotide sequences. The strains were 75·3 to 87·2% identical to one another in complete VP1 nucleotide sequence, but no more than 68% identical in sequence to the prototype strain of any EV serotype. Their complete capsid sequences were closely related to one another, but only distantly related to those of any EV prototype strain. The California and Oman isolates were most closely related to members of EV cluster B, suggesting that they are unclassified members (i.e. a new serotype) of cluster B. The complete genome sequence was determined for one isolate, CA55-1988, and the predicted polyprotein sequence was 86·5 to 89·2% identical to those of other cluster B EV and 56·7 to 61·9% identical to the polyprotein sequences of EV belonging to other clusters. Isolation of this new EV serotype from samples obtained on two continents and over a period of 40 years suggests continued circulation over a wide geographical area. In keeping with standard picornavirus nomenclature, we propose that this new serotype be named ‘enterovirus 73’ (EV73).

We compared the QuickVue Influenza test with PCR for diagnosing pandemic (H1N1) 2009 in 404 persons with influenza-like illness. Overall sensitivity, specificity, and positive and negative predictive values were 66%, 84%, 84%, and 64%, respectively. Rapid test results should be interpreted cautiously when pandemic (H1N1) 2009 virus is suspected.

Rhinovirus is a respiratory virus most typically associated with the common cold and asthma exacerbations, and has not traditionally been considered to play a major role in severe lower respiratory tract infections (LRTIs). As part of a surveillance program for respiratory pathogens of public health importance, children consecutively admitted to intensive care for LRTI at a large tertiary children's hospital were tested with polymerase chain reaction for 11 respiratory viruses and Mycoplasma pneumoniae from February 21 to October 31, 2007; 43 cases were enrolled and rhinovirus was the most frequently detected pathogen, with 21 (49%) positive. Rhinovirus cases frequently were young (median age, 1.4 years [range, 44 days-15 years]), hospitalized for pneumonia (10; 48%), had chronic underlying illnesses (15; 71%), had abnormal chest radiographs (18; 86%), required mechanical ventilation (12; 57%), and had prolonged hospitalization (median length, 7 days [range, 1-29 days]). Coinfection with other viruses or bacteria was common (10; 47%). Rhinovirus may be associated with more severe LRTI in children than previously reported, particularly in the noninfluenza, nonrespiratory syncytial virus season.

Rhinovirus (RV) infections appear to precipitate most asthma exacerbations. To investigate whether RV-16 induces different inflammatory changes in upper and lower airways of asthmatic and healthy subjects, we inoculated 10 nonatopic healthy and 11 atopic asthmatic adults with 2,000 TCID50 RV16. Subjects recorded symptoms and peak flow daily; and they underwent spirometry, methacholine challenge (PC 20 ), nasal lavage, and sputum induction at baseline and on Days 2, 4, 15, and 29 d after inoculation. One asthmatic subject developed an exacerbation requiring prednisone treatment 5 d after inoculation. The cold symptom severity (Jackson score) did not differ between groups. During the cold, asthma symptoms increased slightly from baseline in the asthmatic group; and PC 20 decreased in the healthy group. However, peak flow, bronchodilator use, and spirometry did not change in either group. At baseline, asthmatics had higher neutrophils, eosinophils, and interleukin (IL)-6 in nasal lavage. After inoculation, both groups developed significant increases in nasal neutrophils, IL-6 and IL-8, and modest increases in sputum neutrophils and IL-6, but not IL-8. However, these changes did not differ between groups. IL-5, interferon- g , and RANTES were detected only in nasal lavages from two asthmatic subjects, who had the most severe colds. IL-11 was not detected in any sample. We conclude that inflammatory responses of upper and lower airways during RV-16 colds are similar in asthmatic and healthy subjects, and that RV-16 infection is not by itself sufficient to provoke clinical worsening of asthma. Fleming HE, Little FF, Schnurr D, Avila PC, Wong H, Liu J, Yagi S, Boushey HA. Rhinovirus-16 colds in healthy and in asthmatic subjects: similar changes in upper and lower airways. AM J RESPIR CRIT CARE MED 1999;160:100‐108.

In order to determine the suitability of vaccine strains established in the 1960s for a new vaccine, a comprehensive study of strain variation of adenovirus serotype 4 (AV 4) and AV 7 was undertaken. A 1,500-bp region of the hexon gene containing the AV neutralization epitopes from prototype, vaccine, and community-acquired strains and from wild-type strains from military personnel that cause acute respiratory disease (ARD) was sequenced and analyzed. The whole hexon gene from prototype strains, vaccine strains, and selected isolates was sequenced. AV 7 and AV 7a were found to have distinct genotypes, and all vaccine and wild-type strains recovered from 1963 to 1997 had the AV 7a genotype. There was no significant strain variation in the neutralization epitopes of the AV 7a genotype over a 42-year period. The evolution of AV 4 was more complex, with continuous genetic drift punctuated by replacement with a new strain. The current strain of AV 4, which has been in circulation since 1995, is significantly different from the AV 4 prototype and the vaccine strains. Genetic differences were confirmed to be antigenic differences by neutralization tests, which define the new strain as an AV 4 variant. A type-specific PCR for AV 4, AV 7/7a, and AV 21 was developed, and this PCR facilitated the rapid identification of isolates from outbreaks of ARD.

Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV), as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus) at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus). Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses. © 2013 Yu et al.

Simple Summary In the spring of 2006, four human cases of parapoxvirus infections in Missouri residents were reported to the Centers for Disease Control and Prevention (CDC). We conducted surveys of herders and veterinarians, performed animal and environmental sampling and obtained sera from potential case-patients. We determined that, in general, infected persons may seek advice from veterinarians rather than physicians, thereby giving physicians less clinical experience. The initial perception of increased incidence in Missouri was likely due to reporting bias due to misdiagnosis and increased awareness due to recent publications. Basic personal protective measures are not being routinely utilized. Asymptomatic parapoxvirus infections in livestock may be common and warrants further investigation. Abstract In the spring of 2006, four human cases of parapoxvirus infections in Missouri residents were reported to the Centers for Disease Control and Prevention (CDC), two of which were initially diagnosed as cutaneous anthrax. This investigation was conducted to determine the level of recognition of zoonotic parapoxvirus infections and prevention measures, the degree to which veterinarians may be consulted on human infections and what forces were behind this perceived increase in reported infections. Interviews were conducted and clinical and environmental sampling was performed. Swab and scab specimens were analyzed by real-time polymerase chain reaction (PCR), whereas serum specimens were evaluated for parapoxvirus antibodies. Three case patients were found to have fed ill juvenile animals without using gloves. Forty-six percent of veterinarians reported having been consulted regarding suspected human orf infections. Orf virus DNA was detected from five of 25 asymptomatic sheep. Analysis of extracellular envelope gene sequences indicated that sheep and goat isolates clustered in a species-preferential fashion. Parapoxvirus infections are common in Missouri ruminants and their handlers. Infected persons often do not seek medical care; some may seek advice from veterinarians rather than physicians. The initial perception of increased incidence in Missouri may have arisen from a reporting artifact stemming from heightened concern about anthrax. Asymptomatic parapoxvirus infections in livestock may be common and further investigation warranted.

A seroprevalence survey to recently proposed adenovirus (AV) serotypes AV 48 and AV 49, isolated primarily from AIDS patients, was conducted among the San Francisco Men's Health Study cohort. This cohort of homosexual, heterosexual, or bisexual HIV-seronegative and -seropositive men from selected San Francisco census tracts has been studied since 1984. The presence or absence of type-specific antibody in 628 serum specimens from 1989 was determined by micro-neutralization. Thirty of these subjects (26 positive and four negative) were studied longitudinally. Serum specimens taken at 6-month intervals from 1984 to 1993 were tested to characterize antibody response and to document the advent of these new serotypes. Eight subjects were tested against five other AV serotypes for comparison. AV 48 and AV 49 seroprevalence rates were significantly higher in HIV-seropositives, but infection was not limited to the immunocompromised. Sexual preference was not a significant determinant for AV seroprevalence in HIV-seronegatives. However, the extent and duration of the neutralizing antibody response was strikingly different between homosexuals and heterosexuals: an endemic pattern of continuous reexposure over the 9-year period was seen in 90% of 19 homosexuals, while five of six heterosexuals (83%) had an episodic pattern of exposure with antibody decline to undetectable levels. These data suggest that these viruses may be endemic in some part of the homosexual population and that sexual transmission may be the primary source of continuous reexposure. © 1996 Wiley-Liss, Inc.

The origin of AIDS-associated adenoviruses (AV 43–AV 49) was investigated by examining evolutionary relationships among 18 serologically related subgenus D serotypes and 3 intermediates and determining the mutation rate of a single serotype, AV 48, among clinical isolates from AIDS patients over a 6-year period. Nucleotide sequence of conserved and seven hypervariable regions (HVRs) of the hexon protein, the pVI core protein signal peptide, and noncoding region between the two genes was determined. Among AV 48 isolates the base misincorporation rate was 3.2 per 10,000 bases over 6 years. A 6-bp deletion occurred in one isolate between short direct repeats in HVR 7. Among subgenus D serotypes mutation rates were extremely low in the pVI peptide, the 5′ hexon noncoding region, and first 187 bases of hexon protein. Small 2- and 3-bp deletions between short direct repeats in a polypurine stretch in the noncoding region occurred in 3 strains. Mutation increased with proximity to the HVRs. Within HVR 1, 2, 4, 5, and 7 variability consisted of extensive intrachromosomal illegitimate recombination, including deletions between short direct repeats, insertions and duplications in repetitive polypurine stretches, and numerous base substitutions. All serotypes and intermediates differed by at least one illegitimate recombination event, with one exception. We conclude that AV serotype evolution is driven by illegitimate recombination events (antigenic shift), concommitant with single base mutation (antigenic drift), and that the HVRs are “hot spots” for both. These events could be explained by slippage-misalignment of the AV DNA polymerase in repetitive polypurine stretches during single-strand DNA replication. This mutability in the surface regions of the major viral coat protein confers a distinct survival advantage to this family of viruses.

A microneutralization assay that automates the reading and interpretation of viral infectivity and neutralization data was developed for the characterization of adenoviruses (AV). Virus, serum, and cells were added to 96-well plates, incubated for 7 days, and stained with vital stain. The A550 of solubilized dye was read in a plate reader interfaced to a personal computer which analyzed the results. Correlation of A550 values with visual observation of cytopathic effect was extremely good (r = 0.977625). Clinical isolates of 17 AV from 11 patients were characterized by colorimetric neutralization assay. Prototype AV titers tested were comparable to those determined by tube methods. Prototype homotypic antiserum titers were comparable to or greater than those determined by standard tube neutralization.

Two new adenovirus (AV) subgenus D serotypes, which we propose as AV candidates 48 and 49, are described. Both AV 48 and 49 were unique by serum neutralization and the pattern of restriction endonuclease fragments. By the hemagglutination inhibition assay there was a significant reciprocal cross between AV 48 and 44 and one-way crosses between AV 48 and AV 37 sera and between AV 30 and AV 49 sera. Eleven AV 48 and 5 AV 49 isolates, mainly from AIDS patients, have been identified at the Viral and Rickettsial Disease Laboratory.

A simplified microneutralization procedure is described that uses an empirically determined virus challenge dose, a single dilution of antiserum, and observation of cytopathic effect to determine the adenovirus serotype. The simplified test has faster turnaround time and was 96% concordant with a confirmatory test using serial dilutions of type-specific sera. This method will find utility in high-volume serotyping work.

Background Enterovirus infections are very common and typically cause mild illness, although neonates are at higher risk for severe illness. In 2007, the Centers for Disease Control and Prevention (CDC) received multiple reports of severe neonatal illness and death associated with coxsackievirus B1 (CVB1), a less common enterovirus serotype not previously associated with death in surveillance reports to the CDC. Methods This report includes clinical, epidemiologic, and virologic data from cases of severe neonatal illness associated with CVB1 reported during the period from 2007 through 2008 to the National Enterovirus Surveillance System (NESS), a voluntary, passive surveillance system. Also included are data on additional cases reported to the CDC outside of the NESS. Virus isolates or original specimens obtained from patients from 25 states were referred to the CDC picornavirus laboratory for molecular typing or characterization. Results During 2007-2008, the NESS received 1079 reports of enterovirus infection. CVB1 accounted for 176 (23%) of 775 reported cases with known serotype, making it the most commonly reported serotype for the first time ever in the NESS. Six neonatal deaths due to CVB1 infection were also reported to the CDC during that time. Phylogenetic analysis of the 2007 and 2008 CVB1 strains indicated that the increase in cases resulted from widespread circulation of a single genetic lineage that had been present in the United States since at least 2001. Conclusions Healthcare providers and public health departments should be vigilant to the possibility of continuing CVB1-associated neonatal illness, and testing and continued reporting of enterovirus infections should be encouraged.

During the last couple of years there has been a delightful increase in interest in genetic typing of human rhinoviruses. This is to a large extent due to the discovery of a proposed novel clade, human rhinovirus C (HRV-C). As a consequence, new methods have been reported aiming at unequivocal distinction of traditional HRV prototype strains as well as the newly found uncultivable HRV-C strains. We read with interest the article by Kiang and coworkers (2) describing reverse transcription-PCR-sequencing applications for genetic typing of human rhinoviruses targeting the 5′noncoding region (5′ NCR). A similar approach with largely similar results was published earlier (6). Relatively conserved areas within this region enable broad-spectrum primer design for sensitive methods. However, there are several issues to consider when using the 5′ NCR for genetic typing of HRV. Current taxonomy and classification of picornaviruses are based on capsid region coding sequences. On the basis of this region, a group of previously characterized novel HRV strains form one distinct clade (5, 7) (Fig. ​(Fig.1A),1A), a fact that has also been the basis of the proposal of the Picornavirus Study Group to form a new species, HRV-C, within the Enterovirus genus (4). However, in the article by Kiang et al., on the basis of the partial 5′ NCR sequences, the designated HRV-C strains clustered within the HRV-A clade (2). In contrast, the strains labeled HRV-C in this article formed a clade of their own. As a consequence, because 5′ NCR sequences do not segregate the designated HRV-C from HRV-A (Fig. ​(Fig.1B),1B), they should not be used for typing of new strains. Nevertheless, this region is quite suitable for selected topics of molecular epidemiology, such as analysis of short-term transmission routes (1, 8) or tentative prediction of genetic type as in human enteroviruses (HEV) (9). The sequences nominated as HRV-C by Kiang et al. (2) and by Lee et al. (6) form a new clade in the 5′ NCR. The exact taxonomic position of this clade should be determined according to the clustering of these strains in the capsid region. Clearly, it is divergent from all known HRV and HEV clades in the 5′ NCR, but the decision on whether the strains represent HRV-C or some other picornavirus group cannot be made on the basis of the 5′ NCR alone. FIG. 1. Phylogenetic trees in the VP4/VP2 capsid coding region (A) and in the 5′ NCR (B) of different species of the enterovirus genus showing different clustering of the species in the two regions. Trees were constructed with MEGA4 using the neighbor-joining ... The area close to the beginning of the open reading frame in the 5′ NCR is known to be a recombination hot spot in HEV. Although frequent recombination has not yet been shown for HRV, the analysis of the complete genome sequence data of all HRV prototype strains has not yet been published. Furthermore, the number of completely sequenced genomes of circulating HRV strains has remained low and is too low to conclude that the evolution in the 5′ NCR is always congruent with that of the capsid. Therefore, we would see phylogenetic analysis of the 5′ NCR of HRV as a welcome addition to HRV research, but not a surrogate of capsid coding sequence-based typing.

Adenovirus type 3 (HAdV3) is one of the most prevalent serotypes detected globally. Variants of HAdV3 have been associated with outbreaks of severe disease.To better understand genetic diversity of circulating HAdV3s and examine risk factors for severe disease.Restriction enzyme analysis for genomic characterization of clinical HAdV3 isolates detected by 15 collaborative US laboratories during the period July 2004 to May 2007. Multivariate modeling was employed for statistical analyses.The most common HAdV3 types of 516 isolates studied were HAdV3a2 (36.9%), HAdV3a50 (27.1%), HAdV3a51 (18.0%), and HAdV3a17 (4.6%). Non-HAdV3a genome types were rare (1.2%). HAdV3a50 and HAdV3a51 are newly described variants which became more prevalent in 2006 and 2007 and have been associated with at least one epidemic. Their uniqueness was determined by specific banding profiles generated by digests with endonucleases BclI, BglII, and HindIII. Multivariable risk factor modeling demonstrated that children under 2 years of age (OR=2.7; 95%CI 1.6-4.6), persons with chronic disease (OR=5.1; 95%CI 2.6-9.8), persons infected with HAdV3a2 (OR=3.0; 95%CI 1.5-6.0), with HAdV3a50 (OR=2.5; 95%CI 1.2-5.2), or with multiple or rare strains (OR=2.8; 95%CI 1.3-6.5) were at increased risk of severe HAdV3 clinical disease.In the study period considerable genetic diversity was found among US clinical HAdV3 strains. Novel variants emerged and became prevalent. One such emergent strain may be associated with more severe clinical disease.

Background Human enteroviruses (HEVs) are common pathogens which cause a broad spectrum of illnesses ranging from asymptomatic infection to acute myocarditis and aseptic meningitis. The neutralization assay for serotype determination is labor-intensive and time-consuming. There is a need for a methodology that is more rapid and widely accessible. Objectives Our goals were to develop an algorithm to type enteroviruses which combines both serologic typing, based on indirect immunofluorescence assay (IFA) using type-specific monoclonal antibodies (mAbs) and genotyping, by DNA sequence analysis and to assess the correlation of both IFA and genotyping to traditional viral neutralization by type-specific antisera. Study design Clinical specimens initially determined to be enterovirus positive by nucleic acid detection were grown in cell culture and typed using mAbs. Specimens that could not be typed by mAbs were subject to molecular analysis. Genotyping was performed by a combination of either a primary or semi-nested RT-PCR for a region within VP3/VP1 and followed by direct DNA sequencing of PCR products. Database homology comparisons and phylogenetic analysis were performed based on a defined region (303 nt) within the VP1 gene. Results We inoculated 134 enterovirus nucleic acid amplification-positive specimens into culture and 115 (86%) of these isolates were successfully typed by this algorithm. We have demonstrated a strong correlation between serotyping by viral neutralization to both IFA by type-specific mAbs and genotyping. Conclusions Typing of human enteroviruses can be effectively performed using an integration of antibody-based and molecular methods.

Background. Encephalitis is a relatively rare presentation of enterovirus (EV) infections. Clinical and epidemiologic characteristics of EV encephalitis (EVE) have not been well characterized. Methods. Patients with encephalitis enrolled in the California Encephalitis Project from 1998 to 2005 were tested for a range of pathogens, including EV, using a standardized diagnostic algorithm. EVE was categorized as “confirmed” (EV detected in cerebrospinal fluid [CSF] or brain tissue) or “possible” (EV found in respiratory or fecal specimens or serum EV immunoglobulin [Ig] M detected). We compared clinical and epidemiologic characteristics of EVE with those of other infectious encephalitis cases. Results. EVE was diagnosed in 73 (4.6%) of 1571 patients (45 confirmed cases, 28 possible cases); 11.1% of cases had other infectious causes. Patients with confirmed EVE were younger, although 27% were adults, who presented with significantly less severe symptoms. Serotypes identified in EVE cases correlated with the predominant serotype for the given year reported to the National Enterovirus Surveillance System at the Centers for Disease Control and Prevention. Two of 4 fatal EVE cases were associated with EV71. Conclusion. EVs are an important cause of encephalitis cases requiring hospitalization, in both children and adults. Our data suggest that EVE severity varies by serotype, confirm the importance of CSF/brain tissue polymerase chain reaction, and demonstrate that serum IgM findings are of little value in diagnosing EVE. In the United States, encephalitis causes an estimated 19,000 hospitalizations and up to 1400 deaths each year

Epidemiological data suggest that clinical outcomes of human adenovirus (HAdV) infection may be influenced by virus serotype, coinfection with multiple strains, or infection with novel intermediate strains. In this report, we propose a clinical algorithm for detecting HAdV coinfection and intermediate strains.We PCR amplified and sequenced subregions of the hexon and fiber genes of 342 HAdV-positive clinical specimens obtained from 14 surveillance laboratories. Sequences were then compared with those from 52 HAdV prototypic strains. HAdV-positive specimens that showed nucleotide sequence identity with a corresponding prototype strain were designated as being of that strain. When hexon and fiber gene sequences disagreed, or sequence identity was low, the specimens were further characterized by viral culture, plaque purification, repeat PCR with sequencing, and genome restriction enzyme digest analysis.Of the 342 HAdV-positive clinical specimens, 328 (95.9%) were single HAdV strain infections, 12 (3.5%) were coinfections, and 2 (0.6%) had intermediate strains. Coinfected specimens and intermediate HAdV strains considered together were more likely to be associated with severe illness compared to other HAdV-positive specimens (OR=3.8; 95% CI=1.2-11.9).The majority of severe cases of HAdV illness cases occurred among immunocompromised patients. The analytic algorithm we describe here can be used to screen clinical specimens for evidence of HAdV coinfection and novel intermediate HAdV strains. This algorithm may be especially useful in investigating HAdV outbreaks and clusters of unusually severe HAdV disease.

Increasing recognition of the association of rhinovirus with severe lower respiratory tract illnesses has clarified the need to understand the relationship between specific serotypes of rhinovirus and their clinical consequences. To accomplish this, a specific and sensitive assay to detect and serotype rhinovirus directly from clinical specimens is needed. Traditional methods of serotyping using culture and serum neutralization are time-consuming, limited to certain reference laboratories, and complicated by the existence of over 100 serotypes of human rhinoviruses (HRVs). Accordingly, we have developed a sequence-based assay that targets a 390-bp fragment accounting for approximately two-thirds of the 5′ noncoding region (NCR). Our goal was to develop an assay permitting amplification of target sequences directly from clinical specimens and distinction among all 101 prototype strains of rhinoviruses. We determined the sequences of all 101 prototype strains of HRV in this region to enable differentiation of virus genotypes in both viral isolates and clinical specimens. We evaluated this assay in a total of 101 clinical viral isolates and 24 clinical specimens and compared our findings to genotyping results using a different region of the HRV genome (the VP4-VP2 region). Five specimens associated with severe respiratory disease in children did not correlate with any known serotype of rhinovirus and were found to belong to a novel genogroup of rhinovirus, genogroup C. Isolates were also found that corresponded to the genogroup A2 variant identified in New York and Australia and two other novel group A clusters (GAC1 and GAC2).

Background Adenoviruses are associated with sporadic infection and community and institutional outbreaks; they can cause especially severe disease in infants, young children, immunocompromised persons, and transplant recipients. Fifty-two adenovirus serotypes have been recognized and classified within 7 subgroups or species (A-G), with limited data available on associated clinical syndromes and disease severity in more than one-half of the known serotypes. Methods We describe the clinical presentation and virologic characterization of 1 adult and 2 pediatric patients admitted to 2 separate hospitals during April-May 2006 with severe acute respiratory tract infection. All patients had underlying chronic pulmonary disease; none were severely immunocompromised. All 3 experienced serious chronic sequelae or died. Results Adenovirus was isolated from all 3 case patients. Adenovirus serotype 14, a subspecies B2 serotype not previously associated with severe clinical illness, was confirmed by neutralization assay and sequencing of the hexon gene. Restriction enzyme analysis with BamHI, BglII, HindIII, and SmaI showed all 3 viruses to be identical and to belong to a new genome type that we have designated "Ad14a." Conclusions 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. These case reports highlight the need for rapid diagnosis and improved surveillance, with serotyping and molecular characterization, to identify emerging variants of adenovirus, which may assist with targeted development of antiviral agents or type-specific vaccines.

Author(s): Kistler, Amy; Avila, Pedro C; Rouskin, Silvi; Wang, David; Ward, Theresa; Yagi, Shigeo; Schnurr, David; Ganem, Don; DeRisi, Joseph L; Boushey, Homer A | Abstract: BackgroundBetween 50% and 80% of asthma exacerbations are associated with viral respiratory tract infections (RTIs), yet the influence of viral pathogen diversity on asthma outcomes is poorly understood because of the limited scope and throughput of conventional viral detection methods.MethodsWe investigated the capability of the Virochip, a DNA microarray-based viral detection platform, to characterize viral diversity in RTIs in adults with and without asthma.ResultsThe Virochip detected viruses in a higher proportion of samples (65%) than did culture isolation (17%) while exhibiting high concordance (98%) with and comparable sensitivity (97%) and specificity (98%) to pathogen-specific polymerase chain reaction. A similar spectrum of viruses was identified in the RTIs of each patient subgroup; however, unexpected diversity among human coronaviruses (HCoVs) and human rhinoviruses (HRVs) was revealed. All but one of the HCoVs corresponded to the newly recognized HCoV-NL63 and HCoV-HKU1 viruses, and g20 different serotypes of HRVs were detected, including a set of 5 divergent isolates that formed a distinct genetic subgroup.ConclusionsThe Virochip can detect both known and novel variants of viral pathogens present in RTIs. Given the diversity detected here, larger-scale studies will be necessary to determine whether particular substrains of viruses confer an elevated risk of asthma exacerbation.

Background The human rhinoviruses (HRV) are one of the most common and diverse respiratory pathogens of humans. Over 100 distinct HRV serotypes are known, yet only 6 genomes are available. Due to the paucity of HRV genome sequence, little is known about the genetic diversity within HRV or the forces driving this diversity. Previous comparative genome sequence analyses indicate that recombination drives diversification in multiple genera of the picornavirus family, yet it remains unclear if this holds for HRV. Results To resolve this and gain insight into the forces driving diversification in HRV, we generated a representative set of 34 fully sequenced HRVs. Analysis of these genomes shows consistent phylogenies across the genome, conserved non-coding elements, and only limited recombination. However, spikes of genetic diversity at both the nucleotide and amino acid level are detectable within every locus of the genome. Despite this, the HRV genome as a whole is under purifying selective pressure, with islands of diversifying pressure in the VP1, VP2, and VP3 structural genes and two non-structural genes, the 3C protease and 3D polymerase. Mapping diversifying residues in these factors onto available 3-dimensional structures revealed the diversifying capsid residues partition to the external surface of the viral particle in statistically significant proximity to antigenic sites. Diversifying pressure in the pleconaril binding site is confined to a single residue known to confer drug resistance (VP1 191). In contrast, diversifying pressure in the non-structural genes is less clear, mapping both nearby and beyond characterized functional domains of these factors. Conclusion This work provides a foundation for understanding HRV genetic diversity and insight into the underlying biology driving evolution in HRV. It expands our knowledge of the genome sequence space that HRV reference serotypes occupy and how the pattern of genetic diversity across HRV genomes differs from other picornaviruses. It also reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution.