A Survey of Recent Adenoviral Respiratory Pathogens in Hong Kong Reveals Emergent and Recombinant Human Adenovirus Type 4 (HAdV-E4) Circulating in Civilian Populations

Human adenovirus type 4 (HAdV-E4), which is intriguingly limited to military populations, causes acute respiratory disease with demonstrated morbidity and mortality implications. This respiratory pathogen contains genome identity with chimpanzee adenoviruses, indicating zoonotic origins. A signature of these “old” HAdV-E4 is the absence of a critical replication motif, NF-I, which is found in all HAdV respiratory pathogens and most HAdVs. However, our recent survey of flu-like disease in children in Hong Kong reveals that the emergent HAdV-E4 pathogens circulating in civilian populations contain NF-I, indicating recombination and reflecting host-adaptation that enables the “new” HAdV-E4 to replicate more efficiently in human cells and foretells more potential HAdV-E4 outbreaks in immune-naïve civilian populations. Special attention should be paid by clinicians to this emergent and recombinant HAdV-E4 circulating in civilian populations.

The zoonotic threat potential of adenoviruses is recognized [11], and HAdV-E4 represents the first example. Notably, both the HAdV-E4 prototype and contemporaneous "vaccine" isolates contain a genome signature that is unique to ChAdVs and other SAdVs, i.e., the absence of the Nuclear Factor I (NF-I) binding site that is conserved among the three replication motifs embedded in nearly all HAdV inverted terminal repeats (ITRs) [1]. NF-I is a host transcription factor that binds to nt 23-36 of the HAdV-2 origin of replication [12][13][14][15][16][17][18][19][20] and is recruited by the human adenoviral replication complex [12,17]. It is firmly established as essential through in vitro and in vivo studies (see [18,19] and qtd. in), including replication reconstitution assays, as necessary for efficient adenoviral replication in human cells (see [20] and qtd. in). Recent isolates are recombinants containing this HAdV replication motif [1], presumably permitting an expansion of the virus range into the immune-naïve populations [5][6][7][21][22][23], and should be noted as a molecular evolution example of a post-zoonotic, host-adaptation of a "novel" and "emergent" human pathogen.

Results and Discussion
As noted in the literature, the ITR contains critical conserved DNA replication motifs that include the human transcription factor binding site, NF-I, which is shown to enhance and optimize HAdV replication and growth [2,[12][13][14][15][16][17][18][19][20]. All of the 10 Hong Kong isolates (red) possess this host-adapted ITR, i.e., they have the NF-I binding site that is found in all human respiratory adenoviruses (yellow) (Figure 1). However, this motif is missing in SAdV ITRs (purple) [18], as well as the "old" HAdV-E4 strains (green), e.g., prototype (1952) and "vaccine" (1962) strains, i.e., they have only the core origin and the NF-III motif [1]. The absence of the NF-I motif likely plays a role in limiting the circulation of both SAdVs and prototype HAdV-E4 in human populations, as its acquisition presumably provides efficient and enhanced replication in human cells, i.e., allowed HAdV-E4 to adapt to the new host. This may be the "tipping point" that allows HAdV-E4 entry into the general population that is immunologically-naïve to its epsilon antigen, as HAdV-E4 does not normally circulate outside the U.S. military populations [3,[5][6][7]. This is consistent with reports documenting recombination as an evolution mechanism in the genesis of emergent HAdV pathogens [24], illustrated by HAdV-D53 [25] and HAdV-B55 [24]. HAdV-B55 is a recent major respiratory pathogen in China, after years of quiescence, by virtue of its introduction into immunologically-naïve populations, i.e., without antibodies to its HAdV-B11-like epsilon antigen. Another example is the reemergence of a long-dormant respiratory pathogen HAdV-B14 into an immunologically-naïve population [26].
Consistent with this report, recombination has been reported in SAdVs [27,28] and cross-species transmissions between humans and non-human simians have been reported [28][29][30][31][32][33][34], including TMAdV across "two passages" of human hosts, with clinical manifestations [34], as well as seroprevalence of adenoviruses across species [29,[32][33][34]. These support the hypothesis and data suggesting HAdV-E4 was of zoonotic origins [1,10].  Comparison of HAdV-E4 genomes spanning 65 years reveals highly conserved sequences. For example, the two earliest genomes, isolated within 10 years, are 99.9% identical to each other, differing by 18 mismatches and two insertions (one one-base and one three-base) [35]. One striking difference is a recombination resulting in recent HAdV-E4 genomes acquiring a well-characterized replication-enhancing NF-I motif that distinguishes these isolates from the 1952 prototype and a contemporaneous 1965 co-circulating strain. Although the acquisition of this critical replication motif alone may explain the potentially wider distribution of HAdV-E4, the pair-wise sequence analyses also reveal additional potentially important sequence differences in the E3 gene region [36]. Proteins encoded in the E3 region are involved in interactions with the host immune response and evasion and represent the most variable region between HAdV types [37][38][39]. This report highlights the need to re-explore the understudied E3 genes and their critical roles in the human immune response upon HAdV infection.
However, whether in concert with E3 gene changes or alone, the NF-I acquisition, given "Occam's Razor", may be the critical evolutionary modification and "tipping point" that is necessary for a wider distribution of HAdV-E4 in immune-naïve civilian populations in light of the extensive literature reported for HAdV replication requirements [12][13][14][15][16][17][18][19][20]. This host adaptation may be significant as HAdV-E4 is a highly contagious pathogen with demonstrated morbidity and mortality implications and has now been reported in global civilian populations [6,7,23]. During the preparation of this report, to emphasize further the potential of HAdV-E4 emergence in the global general population, seroepidemiological data identifying HAdV-E4 infections in China and Sierra Leone (Africa) were published [40]. Explicitly, the authors note "An apparent increase in the frequency of human adenovirus type 4 (HAdV-4) infections among general populations has been observed over the past 10 years" [40].

Clinical Specimens and Virus Culture
Nasopharyngeal swab specimens were collected from both outpatients and inpatients who presented with flu-like symptoms, and are archived at Queen Mary Hospital (Hong Kong). Adenoviruses were detected by PCR and were identified further by molecular typing using partial sequence data from the hexon and fiber genes, as previously reported [41]. Then, 10 virus strains that were identified as type 4 were cultured in human lung cell lines (A549), and genomic DNA was extracted using the Viral DNA Extraction Kit from Omega Bio-Tek, Inc. (Norcross, GA, USA), as previously noted [41].

Sequence Data
All sequences are available in GenBank from which whole genome data were retrieved and aligned, and ITR sequences extracted. This resulted in a data set of ITR sequences ranging from 116 to 209 bases, as lengths were variable among the types. A subset of sequences spanning the first 1-66 bases that included the conserved HAdV viral replication sequence motifs were selected for detailed analysis.