You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1. Introduction {#sec0005}
===============

Canine coronavirus (CCoV; order *Nidovirales*, family Coronaviridae) is a large, enveloped, single stranded, RNA virus responsible for enteritis in dogs ([@bib0045]). Recently, due to changes in virus classification, the virus was classified as a member of the genus *Alphacoronavirus*, species *Alphacoronavirus-1*, together with transmissible gastroenteritis virus of swine (TGEV) and feline coronavirus (FCoV) ([@bib0015]). The genome, 27 kb in length, contains two large overlapping open reading frames (ORFs), ORF1a and ORF1b which encompass the 5 two thirds of the genomic RNA and encode polyproteins leading to the replicase complex. The ORFs, encoding for the structural spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins and the non-structural proteins (3a, 3b, 3c, 7a and 7b), are located downstream of the replicase gene ([@bib0045]).

Coronaviruses are characterized by constant genetic evolution and diversity. To date, two different CCoV types have been recognized, CCoV type I (CCoV-I) and CCoV type II (CCoV-II), that share significant genetic similarity with FCoV type I (FCoV-I) and FCoV type II (FCoV-II), respectively ([@bib0045]). Moreover, in 2009, TGEV-like CCoVs of potential recombinant origin were identified and characterized as a new CCoV subtype (CCoV-IIb) ([@bib0050], [@bib0055], [@bib0060]).

CCoV is the causative agent of gastroenteritis in dogs, characterized by high morbidity and low mortality. Clinical signs include anorexia, lethargy, vomiting, mild to severe diarrhoea (usually lasting 1--2 weeks) and occasionally death, mainly in puppies. The disease is more severe in young animals ([@bib0010]). Systemic infections are not usual; however, during the past few years, there have been reports of fatal disease, with CCoV strains detected in the enteric tract, as well as in the organs ([@bib0005], [@bib0050]).

In 2010, CCoV identification, molecular characterization and sequence analysis took place for the first time in Greece, regarding common enteric CCoV-II strains detected in a severe outbreak of diarrhoea in a kennel ([@bib0095]). In the current study we report the quantitation and molecular characterization of two TGEV-like CCoV strains, detected in the organs of two puppies displaying fatal enteritis.

2. Materials and methods {#sec0010}
========================

2.1. Clinical case {#sec0015}
------------------

During summer of 2009, two dead dogs were submitted for laboratory investigation. The dogs were coming from two different pet shops of Thessaloniki, a city of northern Greece. Both dogs, a 6-week-old Yorkshire Terrier (66/09) and a 16-week-old Pomeranian (68/09), presented fever, lethargy, inappetence, severe haemorrhagic diarrhoea and vomiting leading to death, 2 days after the onset of the symptoms. The first puppy was vaccinated with a single dose of a polyvalent vaccine against all major infectious diseases (canine distemper, infectious hepatitis, parvoviral enteritis, parainfluenza and leptospirosis) 2 weeks before the symptoms, while the second one, had never been vaccinated.

Necropsy examination of both dogs revealed linear haemorrhages of the intestinal wall, haemorrhagic enteritis and ulcerated duodenum. Sero-sanguineous fluid was observed in the abdominal cavity of the Pomeranian. Lungs of both puppies were congested with multiple areas of emphysema. No lesions were observed at the heart. Liver of both puppies appeared enlarged, friable and yellow-brown in color with multifocal discolorated spots. Congested vessels in the dura mater of the brain were also observed.

2.2. Screening for viral pathogens {#sec0020}
----------------------------------

Samples from the faeces and the parenchymatous organs were subjected to virological investigations, using methods previously described, regarding common canine viral pathogens e.g., canine parvovirus type 2 (CPV-2) (PCR and real time PCR) ([@bib0020], [@bib0030], [@bib0035]), canine distemper virus (CDV) (RT-PCR) ([@bib0065]), canine adenovirus type 1 and type 2 (CAV-1 and CAV-2) (PCR) ([@bib0075]) and CCoV (RT-PCR) ([@bib0100]).

2.3. Virus isolation {#sec0025}
--------------------

For virus isolation, A-72 cell line (canine fibrosarcoma) was used. The cells were grown in Dulbecco-Minimum Essential Medium (D-MEM) supplemented with 10% foetal bovine serum (FBS). Faecal and tissue samples were homogenized (10%, w/v) in D-MEM and centrifuged at 8000 ×  *g* for 10 min. Supernatants were treated with antibiotics (1000 IU/ml penicillin and 100 μg/ml streptomycin) for 30 min, inoculated on partially confluent A72 cell cultures and then, they were incubated at 37 °C in a 5% CO~2~ incubator. After an adsorption period of 30 min, D-MEM was added. Cells were daily observed for cytopathic effect (cpe) of CCoV for 5 days. An immunofluorescence (IF) assay was used for the detection of CCoV at the infected cells. For the IF assay a 1:100 dilution of cat polyclonal serum specific for *Alphacoronavirus-1* and a 1:100 dilution of goat anti-cat IgG conjugated with fluorescein isothiocyanate (Sigma--Aldrich, USA). Each sample was considered negative after 3 passages.

2.4. CCoV characterization and quantitation {#sec0030}
-------------------------------------------

RNA was extracted from faecal and organ samples of both dogs using the QIAamp Viral RNA Mini Kit and the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), respectively. For CCoV type I and II detection and quantitation in faecal and organ samples, two real time RT-PCR assays with the same sensitivity were used ([@bib0025]). Reverse transcription was performed using GeneAmp^®^ RNA PCR (Applied Biosystems, Italy) according to the manufacturer\'s instructions.

For the discrimination of classical (subtype IIa) and TGEV-like (subtype IIb) CCoVs, two RT-PCR assays with comparable levels of sensitivity were performed, as previously described ([@bib0055]). RT-PCRs with primers 20179/INS-R (CCoV-IIa) or 20179/174-268 (CCoV-IIb) were conducted using SuperScript One-Step RT-PCR for Long Templates (Invitrogen S.R.L.). In order to verify the absence of TGEV strains in the samples that were positive by CCoV-IIb specific assay, an RT-PCR, able to discriminate CCoV and TGEV according to the amplicon size was used ([@bib0120]).

2.5. Sequencing and sequence analysis {#sec0035}
-------------------------------------

The 3′ end of the genome of the CCoV-IIb strains was amplified as previously described, using viral RNA extracted from the lungs, SuperScript One-Step RT-PCR for Long Templates (Invitrogen S.R.L.) and six pairs of primers, specific for overlapping fragments, encompassing ORFs 2, 3a, 3b, 3c, 4, 5, 6, 7a and 7b ([@bib0040]). The nucleotide sequences were determined in both directions by a commercial facility (Beckman Coulter Genomics, United Kingdom). Sequence assembling and analysis were carried out using the BioEdit software package ([@bib0070]) and the National Center for Biotechnology Information (NCBI; [http://www.ncbi.nlm.nih.gov](http://www.ncbi.nlm.nih.gov/)) and European Molecular Biology Laboratory (EMBL; [http://www.ebi.ac.uk](http://www.ebi.ac.uk/)) analysis tools. Phylogenetic analysis was conducted using MEGA4 program ([@bib0110]). Phylogenetic trees, based on the amino acid sequences of S, E, M and N proteins, were elaborated using neighbor-joining method, supplying a statistical support with bootstrapping over 1000 replicates. SimPlot was used for nucleotide sequence comparison of the two strains to *Alphacoronavirus-1* reference strains ([@bib0085]). The sequences of strains 66/09 and 68/09 were registered in GenBank under the accession numbers HQ450376 and HQ450377, respectively.

3. Results {#sec0040}
==========

3.1. CCoV detection, characterization and isolation {#sec0045}
---------------------------------------------------

By means of nested PCR assay for CCoV, viral RNA was detected in faeces, lungs, spleen, kidneys, pancreas, heart, and liver of both puppies. In addition, the brain of the Pomeranian (68/09) was tested positive, while the brain of the Yorkshire Terrier (66/09) was tested negative. By genotype specific real time RT-PCR assays, only CCoV-II was detected in all positive samples. CCoV-II RNA copies/μl of template in the samples are shown in [Table 1](#tbl0005){ref-type="table"} .Table 1CCoV-II RNA copies/μl of template in the samples of the two puppies, tested by genotype-specific real time RT-PCR.Sample66/09 (Yorkshire Terrier)68/09 (Pomeranian)Faeces3.59 × 10^3^7.22 × 10^5^Liver4.64 × 10^4^3.21 × 10^5^Spleen5.20 × 10^5^1.55 × 10^7^Pancreas2.75 × 10^2^2.03 × 10^4^Kidney1.23 × 10^5^3.37 × 10^6^Lung5.99 × 10^6^4.10 × 10^6^Heart1.14 × 10^5^7.08 × 10^6^Brainn.d.2.47 × 10^3^[^1]

In the faecal samples of the two puppies, both CCoV-II subtypes were detected, while in the organs which tested positive, only CCoV which was characterized as TGEV-like (CCoV-IIb) was detected. No TGEV strains were detected in the samples.

The CCoV-IIb strains (66/09 and 68/09) were isolated from the lung homogenates of both puppies. A-72 cells developed a cytopathic effect that consisted of cell rounding and lysis of the monolayer. In addition, cells were tested positive by the immunofluorescence assay. Viral titres on cell cultures were 10^4.25^ (66/09) and 10^4^ TCID~50~/50 μl (68/09) at the 3rd passage.

3.2. Detection of other viral pathogens {#sec0050}
---------------------------------------

Both puppies were tested positive for CPV-2a field strains and negative for CDV, CAV-1 and CAV-2.

3.3. Sequencing results and phylogenetic analysis {#sec0055}
-------------------------------------------------

A total of 8822 and 8828 nucleotides were determined for strains 66/09 and 68/09, respectively, encompassing ORFs 2 (S protein), 3a, 3b, 3c, 4 (E protein), 5 (M protein), 6 (N protein), 7a and 7b. Alignment of the sequences with TGEV, CCoV and FCoV reference strains available in GenBank showed the highest identity to CCoV-IIb reference strain 119/08 (EU924791) (98.2% and 98.9% for 66/09 and 68/09, respectively). The two Greek strains shared an identity of 98%.

The spike protein gene of both strains was 4374 nucleotides long, encoding a protein of 1457 amino acids. When compared to four TGEV-like reference strains (430/07, 119/08, 174/06 and 341/05), no insertions or deletions were observed. The two strains shared 97.6% aa identity to each other, while they showed the highest aa identity to CCoV-IIb reference strain 119/08 (98.3%). By Simplot analysis, the two strains displayed higher nucleotide conservation with the TGEV strain Purdue than with the pantropic CCoV-IIa strain CB/05, at the 5′-end of the S gene ([Fig. 1](#fig0005){ref-type="fig"} ). Phylogenetic analysis revealed that the two Greek strains were more closely related to the four CCoV-IIb reference strains detected in dogs' organs ([Fig. 2](#fig0010){ref-type="fig"}a).Fig. 1S gene sequences analysis with Simplot. The S gene of CCoV-IIb strain 68/09, TGEV strain Purdue and CCoV-IIa pantropic strain CB/05 were plotted against the S gene of CCoV-IIb strain 66/09.Fig. 2Neighbor-joining trees of the Greek strains, based on the S (a), E (b), M (c) and N (d) protein. The trees are rooted on the group 2 canine respiratory coronavirus (CRCoV). The numbers represent the percentage of replicate trees based on 1000 bootstrap replicates.

The envelope protein was found to be 82 amino acids in length, like in most canine coronavirus strains and in three TGEV-like reference strains, 119/08, 174/06 and 341/05, with the exception of 430/07, which is 7 amino acids shorter. The Greek strains had high amino acid identity to each other (98.7%). E protein of strains 66/09 and 68/09 had the highest amino acid identity (100% and 98.7%, respectively) to the CCoV-IIb strains 341/05, 119/08, and to CCoV-IIa CB/05. In the E protein, phylogenetic analysis revealed that the two strains were closely related to CCoV type II strains ([Fig. 2](#fig0010){ref-type="fig"}b).

The membrane protein (M protein) of strains 66/09 and 68/09 was found to be 260 and 262 amino acids long, respectively. Two amino acids were missing from the N-terminal end of the M protein of strain 66/09 in positions 24 and 36, as it has been also observed in reference CCoV-IIb strains 174/06 and 341/05. The two strains shared high amino acid similarity (94.6%). M protein of strains 66/09 and 68/09 had the highest amino acid identity to the CCoV-IIb reference strains detected in the organs (97.3% and 100%, respectively). Phylogenetic analysis of the M protein showed that the two strains were closely related to CCoV-IIa and CCoV-IIb strains ([Fig. 2](#fig0010){ref-type="fig"}c).

The N gene (nucleoprotein) was found to be 1149 nucleotides in length, coding for a polypeptide of 382 amino acids. The two proteins were 98.1% similar. The amino acid sequences had the highest identity with CCoV-IIb 119/08 (98.6% and 99.4%, for 66/09 and 68/09, respectively). Phylogenetic analysis revealed that the two Greek strains were more closely related to CCoV-II reference strains ([Fig. 2](#fig0010){ref-type="fig"}d).

4. Discussion {#sec0060}
=============

Homologous RNA recombination consists one of the major "powers" of genetic evolution and diversity, regarding coronaviruses ([@bib0125]). Under field conditions, mixed infections are required to give rise to recombination events. So far, experimental infections of piglets ([@bib0130]) and dogs ([@bib0080]) with CCoV and TGEV strains, respectively, and the fact that feline aminopeptidase N serves as a functional receptor for both CCoV and TGEV ([@bib0115]), strongly suggest that the two viruses can be found growing at the same "environment" in nature, although the exact host of recombination still remains unknown.

A canine coronavirus strain (UCD-1) of potential recombinant origin with TGEV, was identified for the first time in the late 90s ([@bib0120]). Recently, TGEV-like strains were reported, circulating in dogs in different countries of Europe ([@bib0055]). The strains were detected in faecal samples of dogs with gastroenteritis, they were classified as the new subtype CCoV-IIb and it was suggested that they were a result of recombination events, occurring at different times of these, regarding the old strain UCD-1 ([@bib0055]). In the present study, sequence and phylogenetic analysis takes place for the first time in CCoV-IIb strains detected in Greece.

Moreover, our findings suggest that TGEV-like CCoV strains spreading to the internal organs are circulating in dogs, since so far, there has been only one report in Italy ([@bib0050]). By means of real time RT-PCR, tissue distribution and quantitation of both strains were assessed for the first time, revealing the spreading of the virus to the internal organs. The CPV-2 coinfection may contribute to the spreading of TGEV-like CCoV strains, since so far, they have been only detected in organs of dogs infected also with CPV-2 ([@bib0050]). However, the detection of CCoV-IIa strains strictly in the faeces, in both cases, suggests that CCoV-IIb may have an advantage in disseminating through the dog. In the first report of TGEV-like strains detected in the organs, CCoV-I was also detected strictly in the intestinal content in two cases ([@bib0050]). These cases strongly suggest a difference in pathobiology of CCoV-IIb with respect to CCoV-I/IIa.

By sequence and phylogenetic analysis, it was shown that both strains segregate constantly with the CCoV-IIb reference strains detected in the organs of dogs. Accordingly, the strains were highly similar to TGEV in the 5′ end of the S gene, whereas they clustered with the pantropic CCoV variant CB/05 (subtype CCoV-IIa) in the E, M and N proteins. In a previous study, CCoV-IIb strains detected in the organs were found to share higher amino acid identity with CB/05 than with CCoV common enteric strains, at the level of the same proteins ([@bib0050]). Whether the ability of CCoV-IIb strains to spread to the organs is related to the recently recognized recombinant protein S or to the CB/05-like proteins (E, M and N) needs further research. However, the S-protein "scenario" seems to be more possible, since in coronaviruses S protein mediates receptor attachment, and tissue tropism shift has been associated with mutations in the S gene ([@bib0090]).

In the last decade, new genotypes and subtypes of canine coronavirus have been recognized. Furthermore, a pantropic variant with the ability to cause fatal systemic infection was detected ([@bib0005]). Previous studies revealed that there are antigenic differences between CCoV-I and II ([@bib0105]). In addition, antigenic differences were observed between the two subtypes, CCoV-IIa and CCoV-IIb (TGEV-like CCoVs) ([@bib0050]). Whether the currently circulating vaccines can protect against the TGEV-like recombinant isolates or not has to be verified via vaccinations and experimental infections.

5. Conclusion {#sec0065}
=============

In conclusion, this was the first report of CCoV-IIb tissue distribution. Up to now, there has been only one report of TGEV-like strains detected in internal organs of puppies in Italy. Based on sequence and phylogenetic analysis of the structural proteins, the two Greek isolates were found to be related to the Italian prototype CCoV-IIb strains. In addition, in all cases a mixed infection with CPV-2 was reported. However, the detection of CCoV-IIa strains, strictly at the faeces, suggests that CCoV-IIb strains may have an advantage in disseminating throughout a dog with CPV-2 coinfection, in contrast to common enteric CCoV-IIa strains.

Ntafis Vasileios is grateful to Alexander S. Onassis Public Benefit Foundation for doctoral funding.

[^1]: n.d., not detected.