A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds

Abstract

Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be emphasized in the investigation of disease outbreaks in human and animals.

Figure 1. Illustrative histopathological changes of infected birds.

Panels A–C: Macroscopic and microscopic changes in the liver of bird 1. Panel A: cut surface of liver showing numerous 1–2 mm coalescing necrosis. Panel B: areas of acute coagulative necrosis, which are randomly distributed throughout the liver (hematoxylin and eosin staining; original magnification, ×400). Panel C: Intracytoplasmic accumulations of Chlamydial organisms (arrow) are commonly found throughout the liver parenchyma (gimenez staining; original magnification, ×1000). Panels D–I: immunochemical staining for Psittacine adenovirus HKU1 fiber protein (Original magnification, ×400). The tissue sections were deparaffinized and rehydrated, followed by blocking with 1% bovine serum albumin in PBS to minimize non-specific binding. The sections were incubated with immune serum (panels D, F, G, I) or with non-immune serum (panels E and H) of guinea pigs. After washing three times with PBS, the sections were incubated with FITC-conjugated rabbit anti-guinea pig IgG. For bird 2, apple green fluorescent foci were seen in the cytoplasm of pneumocytes (D) and in the nucleus of hepatocytes (G) with immune serum. As controls, fluorescent foci were not detected in the lung (E) and liver (H) tissue of bird 2 with non-immune serum. For bird 3, fiber protein-positive cells were not detected in the lung (F) and liver (I) tissue.

Figure 2. Correlation of adenovirus viral loads and C. psittaci bacterial loads in lung specimens from Mealy Parrots.

Figure 3. Genome organization of the Psittacine adenovirus HKU1, comparison with duck adenovirus A and fowl adenovirus A.

Figure 4. Bootscan analysis using SimPlot did not show strong phylogenetic signal of recombination in the present adenovirus.

Figure 5. Maximum-likelihood phylogenetic tree showing the relationship of Psittacine adenovirus HKU1 to other adenoviruses inferred from A) hexon protein, B) penton protein and C) polymerase protein, D) fiber-2 protein.

The trees were constructed using PHYML version 3 under the best-fit protein evolution model as selected by ProtTest 3. The bootstrap values were calculated from 1,000 trees. (AdV, adenovirus; BAdV, bovine adenovirus; BtAdV, bat adenovirus; CAdV, canine adenovirus; DAdV, duck adenovirus; FAdV, fowl adenovirus; FrAdV, frog adenovirus; GoAdV, goose adenovirus; MAdV, murine adenovirus; OAdV, ovine adenovirus; PAdV; porcine adenovirus; PiAdV, pigeon adenovirus; RAdV, raptor adenovirus; SAdV, simian adenovirus; SnAdV, snake adenovirus; SPSAdV; south polar skua adenovirus; TAdV; turkey adenovirus).