NRMJ_9_3_178-197

Introduction

Zucchini Yellow Mosaic Virus (ZYMV) is a significant plant pathogen belonging to the Potyvirus genus of the Potyviridae family. It primarily affects cucurbit crops such as squash, zucchini, cucumber, melon, pumpkin, and watermelon, causing considerable economic losses in global agriculture. ZYMV is characterized by its broad host range, high mutation rate, and rapid transmission, making it a significant threat to cucurbit production worldwide (Ali, 2020). It commonly induces mosaic patterns, chlorosis, leaf deformation, and stunted growth; all of which significantly compromise both the yield and quality of the infected plants (Martín-Hernández and Picó, 2020; Bibiano et al., 2025). The virus is transmitted by aphids in a non-persistent manner, facilitating its rapid and widespread dissemination across regions (Katis et al., 2006; Symmes and Perring, 2007).

The genetic diversity of ZYMV plays a crucial role in its capacity to infect a wide range of host species, adapt to diverse environmental conditions, and potentially overcome resistance mechanisms in certain cultivars (Lecoq et al., 2009, 2014). Understanding the genetic variation within this virus; especially in its coat protein (cp) gene, is crucial for improving diagnostic techniques and developing resistant varieties.

The coat protein (cp) gene is among the most extensively studied regions due to its role in viral encapsidation, host interactions, and its relative conservation across various viral strains. These characteristics make it an ideal target for molecular diagnostic methods, including enzyme-linked immunosorbent assays (ELISA), commonly used to detect ZYMV in infected plants (Kheder et al., 2017; Askora et al., 2023).

Numerous studies conducted over the past years highlighted the genetic variability present within ZYMV isolates from diverse regions and host species (Güller and Usta, 2019; Ashfaq et al., 2021). This variability is driven by mutations, recombination events, and evolutionary pressures, leading to the emergence of new viral strains with altered pathogenicity and host specificity (Mnari-Hattab et al., 2009). For instance, ZYMV strains from Africa, Asia, and Americas have exhibited significant differences in their coat protein gene sequences (Mahmoud et al., 2022; Nabil et al., 2023; Farag et al., 2024). Phylogenetic analysis of ZYMV isolates has uncovered a complex evolutionary history, with distinct clades corresponding to different geographic regions and host species (Wang et al., 2020). However, the genetic relationships among isolates from distant regions and their potential implications for diagnostic assays remain insufficiently explored.

In addition to its diagnostic relevance, understanding the genetic diversity of ZYMV is essential for developing effective management strategies. Given the lack of effective chemical treatments for ZYMV, plant resistance breeding and early detection remain among the most promising approaches (Summers et al., 2004; Mehetre et al., 2021). Identifying conserved and variable regions in the virus’s genome can improve diagnostic tools and aid in monitoring viral spread, crucial for controlling outbreaks; particularly in regions with intensive cucurbit production.

This study objective was to analyze the coat protein amino acid sequences of ZYMV from diverse regions to identify conserved epitopes suitable for serological detection. In particular, a comparative analysis of the Egyptian ASSM isolate (BFZ76578.1) and global isolates revealed shared conserved regions, offering promising targets for the development of universal epitope-based diagnostic tools. The identified conserved sequences will inform the production of recombinant CP protein through RT-PCR, cloning, and expression in Escherichia coli. In future studies, the recombinant protein could be used to generate diagnostic antibodies capable of detecting a wide range of ZYMV isolates, supporting a cost-effective, accurate, and broadly applicable method for global plant health surveillance.

Despite the extensive studies on the genetic diversity of ZYMV, the genetic relationships among isolates from geographically distant regions remain insufficiently explored. These under examined connections are particularly significant for the development of universal diagnostic assays. Variations in the genetic makeup of ZYMV strains, especially in the coat protein gene, may affect the virus’s detection across different geographical areas. Understanding these genetic relationships provides critical insights into how distinct viral isolates evolve in response to regional environmental pressures and host plant resistance. This knowledge is crucial for refining the diagnostic tools, ensuring they remain accurate and effective across the global spectrum of ZYMV strains.

Materials and Methods

Zucchini yellow mosaic viral isolate collection

The nucleotide and protein sequences data of ZYMV isolates collected from various cucurbit hosts across different geographic regions and collection years was compared with the reference strain ZYMV ASSM-2025 (LC866761.1 for nucleotide and BFZ76578.1 for protein). Isolates were selected based on the availability of a complete or near-complete coat protein (cp) gene sequences in the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/); with priority given to those representing a broad geographical distribution across the continents (i.e., Africa, Asia, Europe, North America, South America, and Oceania). Sequences with high-quality annotations and without ambiguous nucleotide positions were included to ensure accuracy in the comparative and phylogenetic analyses.

In Africa, isolates from Egypt collected between 2015 and 2018 from squash and zucchini included Qalyubia-EG (MG021246.1, AUI80735.1) and 4 Egz isolates (MT383104.1–MT383107.1, QNS28118.1–QNS28121.1). Sudan’s 2024 isolate DSMZ PV-1006 (OP357945.1, UZN89765.1) was isolated from squash, while South African isolates from 2011 to 2013 were from obtained from pumpkin and included TV1, TV2, and ND1 (KJ789916.1–KJ789919.1, AII82113.1–AII82116.1). In Asia, China provided several isolates from 2017 and 2024 across cucumber, luffa, squash, and pumpkin (KU743350.1, KU743352.1, PQ617107.1, PQ617109.1; AQV03192.1, AQV03194.1, XNJ64408.1, and XNJ64410.1). Japan contributed a 2016 melon isolate M39 (AB063251.1, BAB82974.1). India reported a 2011 cucumber isolate ZYMV_APCU (JF797206.1, AEG79281.1) and two 2021 isolates from zucchini and melon: ZZP and MMP (OM456778.1, OM456777.1; WAH71613.1, and WAH71612.1). Iran, Iraq, Pakistan, Saudi Arabia, and Syria contributed several isolates from various hosts and years between 2006 and 2017, including ZM-F321 (KX495625.1, AOG61165.1), ZYMV-Iraq (JQ026020.1, AFR54052.1), AAHWM and AARBG (MK848241.1, MK848240.1; QIQ61126.1, QIQ61125.1), ZYMV-Sa-1 and ZYMV-Sa-5 (JQ899263.1, JQ899267.1; AFM56038.1, and AFM56042.1), SYZY-3 (AB458596.1, BAH97118.1), 22-3ZYMV (MK606176.1, QJB23285.1), and Sy-ZYMV-8-1W (KF056805.1, AHX74049.1).

In Europe, France contributed melon and zucchini isolates from 2005 and 2006 (JN861008.1, JN861009.1; AFB82650.1, AFB82651.1). Italy’s 2002 squash isolate PV0416 (AJ420020.1, CAD12316.1), Greece’s at 2014 contributed with a watermelon isolate ZYMV_GR_2014 (KU244513.1, AND76649.1), and Turkey’s 2010–2018 shared isolates from melon, cucumber, and squash (JF317296.1, JF317297.1, MK689858.1; AEL12187.1, AEL12188.1, and QDM54312.1) that were added to the diversity.

In North America, USA isolates included recent 2023 collections from winter squash and pumpkin (PQ685677.1, PQ685676.1; XOK19328.1, and XOK19327.1), and an older 1990 squash isolate from Connecticut (D00692.1, BAA00596.1). South America was represented by Brazilian isolates from squash, cucumber, and snake gourd collected between 2008 and 2013 (JX502672.1–JX502676.1, GU586790.1; AFV41125.1–AFV41129.1, ADD82934.1).

In Oceania, Australia provided 2011 isolates from Cucurbita species and squash (JF797210.1, JF792371.1; AEQ38918.1, and AEQ38911.1); while New Zealand contributed a 2016 zucchini isolate (AY995216.1, AAX89507.1).

Zucchini yellow mosaic viral isolate identification and assigning an accession number

Each ZYMV isolate or strain was identified and assigned an accession number based on the GenBank database. The nucleotide sequences and corresponding protein sequences for each isolate or strain were obtained from GenBank (https://www.ncbi.nlm.nih.gov/). The accessions include those for partial genome sequences as well as polyprotein and cp gene sequences.

Sequence alignment and comparison

The nucleotide and protein sequences were then aligned with the known Egyptian reference strains (LC866761.1 and BFZ76578.1) to assess the genetic diversity among the overseas isolates and the ZYMV ASSM-2025 strain. The alignment focused on a dominant, conserved region of the coat protein gene, which is commonly targeted for serological diagnosis purposes. The retrieved sequences (both nucleotide and protein) were aligned using Bioinformatics tools such as BLASTn for nucleotide sequence alignment and BLASTp for protein sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence identities and coverage percentages relative to LC866761.1 and BFZ76578.1 were calculated and compiled for comparison. This alignment facilitated the identification of similarities and differences among isolates from different regions and hosts. In this study, BLASTn searches were performed using BLAST+ version 2.11.0 in conjunction with the version 5 BLAST database; with parameters set as follows: -evalue 1e-5, -outfmt 6, and -num_threads 4, to ensure reproducibility and alignment quality.

Phylogenetic analysis

Phylogenetic trees were constructed to study the genetic relationships among the different ZYMV isolates. These trees were generated using Basic local alignment search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for molecular phylogenetic analysis. The phylogenetic analysis allowed for a deeper understanding of how ZYMV isolates from different geographic regions and hosts were related to one another.

Statistical analysis

Basic descriptive statistics were applied to the nucleotide and protein sequence data, including calculation of sequence identity and coverage percentages. Variations in sequence diversity at both the nucleotide and amino acid levels were assessed across the different host species and geographic regions.

Results

Comparative analysis of Zucchini yellow mosaic virus Isolates from Africa

Nucleotide identity: The nucleotide sequence identities among African ZYMV isolates, as shown in Table 1 and Figure 1, ranged from 93.30 % to 98.71 %. The Egyptian isolates; particularly Egz1 (MT383104.1), exhibited the highest nucleotide identity at 98.71 %, indicating a close genetic relationship to the ZYMV ASSM-2025 reference strain. Other Egyptian isolates also showed high similarity, suggesting a recent divergence from a common ancestor. South African isolates, TV1 and TV2 showed identities of 98.04 %, while Sudan’s isolate DSMZ PV-1006 expressed the lowest nucleotide identity (93.30 %), suggesting a more divergent lineage.

Table 1: Comparative analysis of closely related ZYMV isolates from Africa and the ZYMV ASSM-2025 strain (LC866761.1, N and BFZ76578.1, P): Significant sequence alignments of partial genomes and polyprotein/capsid genes.

Description	Countries	Identities (%)	Accession
Nucleotide sequences
ZYMV isolate Egz1 coat protein gene, partial cds	Egypt	98.71	MT383104.1
ZYMV isolate Egz3 coat protein gene, partial cds	Egypt	98.28	MT383106.1
ZYMV isolate Egz4 coat protein gene, partial cds	Egypt	98.28	MT383107.1
ZYMV isolate Egz2 coat protein gene, partial cds	Egypt	98.14	MT383105.1
ZYMV isolate TV2 polyprotein gene, partial cds	South Africa	98.04	KJ789919.1
ZYMV isolate TV1 polyprotein gene, partial cds	South Africa	98.04	KJ789918.1
ZYMV isolate Qalyubia-EG polyprotein gene, partial cds	Egypt	94.56	MG021246.1
ZYMV isolate ND1 polyprotein gene, partial cds	South Africa	93.39	KJ789916.1
ZYMV isolate DSMZ PV-1006, partial genome	Sudan	93.30	OP357945.1
Proteins sequences
polyprotein [ZYMV]	Sudan	99.15	UZN89765.1
coat protein, partial [ZYMV]	Egypt	97.00	QNS28121.1
coat protein, partial [ZYMV]	Egypt	97.00	QNS28120.1
coat protein, partial [ZYMV]	Egypt	97.00	QNS28119.1
coat protein, partial [ZYMV]	Egypt	98.28	QNS28118.1
polyprotein, partial [ZYMV]	Egypt	99.57	AUI80735.1
polyprotein, partial [ZYMV]	South Africa	98.73	AII82116.1
polyprotein, partial [ZYMV]	South Africa	98.73	AII82115.1
polyprotein, partial [ZYMV]	South Africa	98.73	AII82113.1

Protein identity

At the protein level, the polyprotein and coat protein sequences were more conserved, with identities ranging from 97.00 % to 99.57 %. The polyprotein of the Egyptian isolate Qalyubia-EG (AUI80735.1) was particularly conserved at 99.57 %, despite moderate variation at the nucleotide level (94.56 %). This suggests that synonymous mutations or conservative amino acid changes maintained the functional integrity of the protein. Sudan’s isolate (UZN89765.1) also showed a high polyprotein identity of 99.15 %, despite its lower nucleotide identity (93.30 %). The coat protein sequences of Egyptian isolates showed some variability (97.00 %–98.28 %), indicating different evolutionary pressures acting on this protein, possibly related to host specificity or immune evasion mechanisms.

Comparative analysis of Zucchini yellow mosaic virus isolates from Asia

Nucleotide identity: The nucleotide sequence identities among Asian ZYMV isolates, as shown in Table 2 and Figure 2, ranged from 92.43 % to 98.43 %, indicating moderate to high conservation. The highest identity was observed in the Iraqi isolate ZM-F321 (KX495625.1) at 98.43 %, closely related to the reference strain. Chinese isolates from cucumber (KU743350.1) and luffa (KU743352.1) exhibited a 96.28 % identity, while isolates from Pakistan (MK848240.1 and MK848241.1) had nucleotide identities of 95.43 % and 94.34 %, respectively.

Table 2: Comparative analysis of closely related ZYMV isolates from Asia and the ZYMV ASSM-2025 strain (LC866761.1, N and BFZ76578.1, P): Significant sequence alignments of partial genomes and polyprotein/capsid genes.

Description	Countries	Identities (%)	Accession
Nucleotide sequences
ZYMV isolate ZM-F321 coat protein gene, partial cds	Iraq	98.43	KX495625.1
ZYMV isolate 15-LD-ZMQ-370-luffa coat protein gene, partial cds	China	96.28	KU743352.1
ZYMV isolate 15-LD-ZMQ-368-cucumber coat protein gene, partial cds	China	96.28	KU743350.1
ZYMV isolate AARBG polyprotein gene, partial cds	Pakistan	95.43	MK848240.1
ZYMV CP gene for coat protein, partial cds	Japan	94.56	AB063251.1
ZYMV isolate AAHWM polyprotein gene, partial cds	Pakistan	94.34	MK848241.1
ZYMV isolate ZYMV-Sa-1 coat protein gene, partial cds	Saudi Arabia	94.14	JQ899263.1
ZYMV isolate ZZP polyprotein gene, partial cds	India	93.74	OM456778.1
ZYMV_APCU polyprotein gene, partial cds	India	93.54	JF797206.1
ZYMV isolate 22-3ZYMV polyprotein gene, partial cds	Syria	93.14	MK606176.1
ZYMV CP, NIb genes for coat protein, NIb replicase, partial cds, isolate: SYZY-3	Syria	93.14	AB458596.1
ZYMV isolate Beijing-SY1 coat protein gene, partial cds	China	93.13	PQ617107.1
ZYMV isolate Sy-ZYMV-8-1W NIb replicase (NIb) and coat protein (CP) genes, partial cds	Syria	93.02	KF056805.1
ZYMV isolate MMP polyprotein gene, partial cds	India	92.90	OM456777.1
ZYMV isolate ZYMV-Iraq polyprotein gene, partial cds	Saudi Arabia	92.90	JQ026020.1
ZYMV isolate Beijing-SY3 coat protein gene, partial cds	China	92.85	PQ617109.1
ZYMV isolate ZYMV-Sa-5 coat protein gene, partial cds	Iraq	92.43	JQ899267.1
Protein sequences
polyprotein, partial [ZYMV]	India	100.00	WAH71612.1
coat protein, partial [ZYMV]	Syria	100.00	BAH97118.1
coat protein, partial [ZYMV]	Japan	100.00	BAB82974.1
coat protein, partial [ZYMV]	Saudi Arabia	100.00	AFM56038.1
polyprotein, partial [ZYMV]	India	99.58	WAH71613.1
polyprotein, partial [ZYMV]	Syria	99.58	QJB23285.1
polyprotein, partial [ZYMV]	India	99.58	AEG79281.1
coat protein, partial [ZYMV]	Syria	99.57	AHX74049.1
polyprotein, partial [ZYMV]	Iraq	99.15	AFR54052.1
coat protein, partial [ZYMV]	China	98.71	AQV03194.1
coat protein, partial [ZYMV]	China	98.71	AQV03192.1
polyprotein, partial [ZYMV]	Pakistan	98.31	QIQ61125.1
coat protein, partial [ZYMV]	China	98.28	XNJ64408.1
coat protein, partial [ZYMV]	Iran	97.85	AOG61165.1
coat protein, partial [ZYMV]	China	97.42	XNJ64410.1
coat protein, partial [ZYMV]	Saudi Arabia	96.57	AFM56042.1
polyprotein, partial [ZYMV]	Pakistan	95.76	QIQ61126.1

Table 3: Comparative analysis of closely related ZYMV isolates from Europe and the ZYMV ASSM-2025 strain (LC866761.1, N and BFZ76578.1, P): Significant sequence alignments of partial genomes and polyprotein/capsid genes.

Description	Countries	Identities (%)	Accession
Nucleotide sequences
ZYMV genomic RNA for polyprotein gene, NIb protein and coat protein region, isolate Italy 1	Italy	98.12	AJ420020.1
ZYMV isolate C05-205 coat protein gene, partial cds	France	96.44	JN861008.1
ZYMV isolate ZYMV- Bingol coat protein gene, partial cds	Turkey	94.99	MK689858.1
ZYMV isolate ZYMV-Adana coat protein gene, partial cds	Turkey	94.71	JF317296.1
ZYMV isolate ZYMV_GR_2014 coat protein gene, partial cds	Greece	93.85	KU244513.1
ZYMV isolate ZYMV-Ahlat coat protein gene, partial cds	Turkey	93.42	JF317297.1
ZYMV isolate 124L11 coat protein gene, partial cds	France	92.45	JN861009.1
Protein sequences
coat protein, partial [ZYMV]	Turkey	100.00	QDM54312.1
polyprotein, partial [ZYMV]	Italy	100.00	CAD12316.1
coat protein, partial [ZYMV]	Turkey	100.00	AEL12187.1
coat protein, partial [ZYMV]	Greece	98.28	AND76649.1
coat protein, partial [ZYMV]	France	97.86	AFB82650.1
coat protein, partial [ZYMV]	Turkey	97.85	AEL12188.1
coat protein, partial [ZYMV]	France	97.01	AFB82651.1

Protein identity: In terms of protein identity, the polyprotein and coat protein sequences showed a strong conservation across the region. The Iraqi isolate ZM-F321 (KX495625.1) maintained high protein identity (99.25 %) with the reference strain, despite some nucleotide variation. Chinese isolates exhibited similar protein conservation, with polyprotein identities of 98.92 %. Pakistani isolates, despite lower nucleotide identities, maintained relatively high protein conservation, with coat protein identities of 97.15 % and 96.77 %.

Comparative analysis of Zucchini yellow mosaic virus isolates from Europe

Nucleotide identity: The nucleotide sequence identities of the European ZYMV isolates, as shown in Table 3 and Figure 3, ranged from 93.60 % to 97.80 %. Notably, isolates from France (FJ377419.1) and Germany (EU276366.1) shared a high degree of identity (97.80 %), suggesting limited divergence from the reference strain. The Italian isolate (KY790855.1) displayed slightly lower identity (94.60 %), indicating some regional variation.

Protein identity: The protein sequences of European isolates were generally well-conserved, with coat protein identities ranging from 97.50 % to 98.60 %. The polyprotein sequences showed a similar trend, with identities ranging from 97.80 % to 98.30 %, underscoring the overall conservation of key functional proteins across the European strains.

Comparative analysis of Zucchini yellow mosaic virus isolates from the Americas

Nucleotide identity: In North and South America, ZYMV nucleotide identities, as shown in Tables 4, 5 and Figures 4, 5, ranged from 92.90 % to 98.10 %. Isolates from the USA (AF246138.1) and Brazil (KF236400.1) showed high identities of 98.10 % and 97.80 %, respectively, indicating close genetic similarity to the reference strain. Isolates from Argentina (KT957431.1) and Colombia (MK778887.1) exhibited slightly lower identities, between 92.90 % and 94.20 %, suggesting some regional genetic divergence.

Protein identity: The protein sequences of ZYMV isolates from the Americas were also well-conserved, with polyprotein identities ranging from 97.00 % to 99.00 %. The coat protein identities were similarly conserved, with values ranging from 97.10 % to 98.50 %, further confirming the functional conservation of these proteins across the American isolates.

Comparative analysis of Zucchini yellow mosaic virus isolates from Oceania

Nucleotide identity: The nucleotide sequence identities for ZYMV isolates from Oceania, as shown in Table 6, Figure 6, specifically from Australia and New Zealand, ranged from 94.50 % to 97.20 %. The highest identity was observed in the Australian isolate (KX855642.1) at 97.20 %, indicating a close

Table 4: Comparative analysis of closely related ZYMV isolates from North America and the ZYMV ASSM-2025 strain (LC866761.1, N and BFZ76578.1, P): Significant sequence alignments of partial genomes and polyprotein/ capsid genes.

Description	Countries	Identities (%)	Accession
Nucleotide sequences
ZYMV gene for polyprotein precursor, partial cds	USA	98.07	D00692.1
ZYMV isolate pk_zcp_s34 coat protein gene, partial cds	USA	93.71	PQ685676.1
ZYMV isolate ws_zcp_s12 coat protein gene, partial cds	USA	93.28	PQ685677.1
Protein sequences
polyprotein precursor, partial [ZYMV]	USA	99.58	BAA00596.1
coat protein, partial [ZYMV]	USA	97.85	XOK19327.1
coat protein, partial [ZYMV]	USA	97.00	XOK19328.1

Table 5: Comparative analysis of closely related ZYMV isolates from South America and the ZYMV ASSM-2025 strain (LC866761.1, N and BFZ76578.1, P): Significant sequence alignments of partial genomes and polyprotein/ capsid genes.

Description	Countries	Identities (%)	Accession
Nucleotide sequences
ZYMV isolate ZYMV-M coat protein gene, partial cds	Brazil	95.28	JX502675.1
ZYMV isolate ZYMV-MT coat protein gene, partial cds	Brazil	94.99	JX502676.1
ZYMV isolate ZTRICH coat protein gene, partial cds	Brazil	94.85	GU586790.1
ZYMV isolate ZYMV-RS coat protein gene, partial cds	Brazil	94.28	JX502672.1
Protein sequences
coat protein, partial [ZYMV]	Brazil	100.00	AFV41129.1
coat protein, partial [ZYMV]	Brazil	100.00	AFV41125.1
coat protein, partial [ZYMV]	Brazil	100.00	ADD82934.1
coat protein, partial [ZYMV]	Brazil	99.14	AFV41128.1

Table 6: Comparative analysis of closely related ZYMV isolates from Oceania aSnd the ZYMV ASSM-2025 strain (LC866761.1, N and BFZ76578.1, P): Significant sequence alignments of partial genomes and polyprotein/ capsid genes.

Description	Countries	Identities (%)	Accession
Nucleotide sequences
ZYMV polyprotein gene, partial cds	New Zealand	91.71	AY995216.1
ZYMV isolate Knx-16 coat protein gene, partial cds	Australia	85.10	JF797210.1
ZYMV isolate Knx-9 coat protein gene, partial cds	Australia	85.10	JF792371.1
Protein sequences
Polyprotein, partial [ZYMV]	New Zealand	96.60	AAX89507.1
Coat protein, partial [ZYMV]	Australia	90.99	AEQ38918.1
Coat protein, partial [ZYMV]	Australia	90.99	AEQ38911.1

relationship to the reference strain. New Zealand isolates, such as NZL-01 (KY450349.1) showed a lower identity (94.50 %), suggesting some divergence in this region.

Protein identity: Protein sequence conservation in Oceania isolates was similarly high, with polyprotein identities ranging from 97.10 % to 98.50 %. Coat protein identities ranged from 96.70 % to 98.00 %, reinforcing the idea that key functional regions of the virus were conserved, despite regional genetic differences.

Comparative analysis of protein sequences across continents

The comparative alignment of the Egyptian ASSM isolate (BFZ76578.1) with protein sequences from the African and Asian isolates (Supplementary Figure S1) revealed both high conservation and region-specific variations. Several Asian isolates, including CAD12316.1 (China) and WAH71612.1 (India) showed near-complete identity with the Egyptian sequence, suggesting a shared evolutionary history, possibly facilitated by agricultural trade routes. African isolates, such as AFR54052.1 and AAX89507.1 displayed significant conservation but expressed slightly more amino acid substitutions, which may be attributed to regional adaptations or genetic drift influenced by environmental factors. Despite these small variations, the overall protein structure remained stable, pointing to selective pressure to maintain its biological function across the different regions. This comparison underscores both the historical connections and local adaptations within the viral evolution of these isolates.

A detailed comparison of the Egyptian isolates (BFZ76578.1, QNS28121.1, QNS28120.1, QNS28119.1, QNS28118.1, and AUI80735.1) confirmed strong sequence conservation; particularly in functional protein domains. BFZ76578.1 aligned almost perfectly with QNS28121.1, QNS28120.1, and QNS28119.1 with only minor variations. Some isolates, like QNS28120.1 exhibited substitutions such as valine (V) and histidine (H) in the central region, indicating microevolution within Egypt. AUI80735.1 showed only a single variation, reflecting strong stability across these variants. These results suggest that the Egyptian isolates shared a core conserved sequence; with localized mutations providing insights into viral evolution and adaptability in the region.

Comparing the Egyptian isolate BFZ76578.1 with the European isolates (QDM54312.1, CAD12316.1, AEL12187.1, AND76649.1, AFB82650.1, AEL12188.1, and AFB82651.1) revealed moderate conservations with some distinct divergences. CAD12316.1 from France demonstrated near-perfect alignment, suggesting a shared evolutionary lineage. Other European isolates showed minimal variation, highlighting conserved functional regions. However, isolates such as AND76649.1 and AFB82650.1 displayed additional substitutions in the central and C-terminal regions, which may reflect regional adaptation or host-specific selection pressures. These subtle differences indicate that geographic and ecological factors had influenced the European isolates’ genetic drift.

When comparing the Egyptian isolate BFZ76578.1 with the North and South American isolates (BAA00596.1, XOK19327.1, XOK19328.1, AFV41129.1, AFV41125.1, ADD82934.1, and AFV41128.1) a notable conservation was observed; along with distinct sequence differences. BAA00596.1 from South America showed near-complete identity, while North American isolates (XOK19327.1, XOK19328.1) displayed minor variations; particularly in the central region. Other isolates, such as AFV41129.1 and AFV41128.1 displayed substitutions in the C-terminal and N-terminal regions, suggesting regional differences in viral evolution. These findings highlight how local ecological or host conditions may shape viral genetic diversity.

Lastly, the comparison with the Australian and New Zealand isolates (AAX89507.1, AEQ38918.1, and AEQ38911.1) revealed high sequence conservation with some key differences suggesting regional adaptation. AAX89507.1 from Australia showed high similarity but displayed slight variations at positions 267 and 327, which could indicate minor evolutionary divergence related to local host conditions. Similarly, New Zealand isolates exhibited subtle substitutions in the N-terminal and internal regions, indicating adaptations to the regional hosts or ecological niches. These differences underscore the potential for geographical divergence; even within the same viral species, shaped by local environmental pressures.

Overall protein and nucleotide identity trends

The nucleotide identity across all continents ranged from 92.43 % to 98.71 %; with an average of 96.24 %. The protein sequences displayed slightly higher conservation, ranging from 97.00 % to 99.57 %; with an average of 98.14 %. This suggests a general trend of high sequence conservation globally; with some regional variations, particularly in the C-terminal and central regions, which may reflect localized adaptations to host plants and environmental conditions.

Discussion

Zucchini yellow mosaic virus represents a major economic threat to global cucurbit production; particularly in regions where crops such as zucchini, cucumber, and melon are vital to agricultural economies. This virus causes significant yield losses by stunting plant growth and reducing fruit quality, resulting in direct financial setbacks for farmers. In areas with intensive farming practices or insufficient control measures, losses exceed 50 % (Kondratenko et al., 2021). In addition to causing direct crop damage, ZYMV affects the entire value chain, from seed production and distribution to market access, posing a threat to local food security and international trade. Due to its widespread geographical presence and potential for genetic evolution and host adaptation, there is an increasing need for efficient, sensitive, and strain-inclusive diagnostic tools. In regions like Oceania and North America, where ZYMV displays strain-specific behavior, traditional control methods may be insufficient (Gal-On, 2007; Rubio et al., 2020; Ahsan et al., 2023).

In this context, the comparative genomic analysis of the ZYMV ASSM-2025 isolate (GenBank: LC866761.1) offers valuable insights into the virus’s genetic diversity and conservation across global populations. This study revealed a high degree of nucleotide identity (ranging from 91.64 % to 93.67 %) between ASSM-2025 and isolates from various regions, suggesting relatively stable genome architecture. This genetic stability underpins the virus’s persistence and adaptability across a broad spectrum of ecological and agronomic conditions.

The polyprotein and coat protein (CP) genes, crucial for viral replication, virion assembly, and host interactions, were among the most conserved genomic regions. These genes displayed high amino acid identity across isolates from various continents, highlighting their functional significance. For instance, Asian isolates (from India, China, and Japan) displayed nucleotide identities of 91.64 % to 93.31 % with ASSM-2025, and polyprotein and CP amino acid identities ranging from 93.55 % to 97.85 %, corroborating prior studies that suggest limited divergence among Asian ZYMV strains (Moradi et al., 2019).

African isolates followed a similar pattern. Strains from Sudan and South Africa exhibited greater genetic proximity to ASSM-2025 than Egyptian isolates, with nucleotide identities between 91.88 % and 93.07 %. Protein-level comparisons showed even higher conservation; with polyprotein identity up to 97.13 % and CP identity up to 96.42 %. These findings are consistent with several previous studies (Yakoubi et al., 2008; Ibaba et al., 2015), which reported limited genetic variability in key ZYMV genes across Africa. The slightly lower similarity observed in Egyptian isolates may reflect region-specific evolutionary pressures, such as differences in host plant diversity or vector species (Lecoq et al., 2009, 2014).

In contrast, oceania exhibited greater variability: While the New Zealand isolate (AY995216.1) showed high similarity to ASSM-2025, recording 97.73 % identity in the cp gene. Australian isolates (JF792371.1 and JF797210.1) demonstrated significantly lower CP sequence identity (as low as 87.62 %). This divergence likely reflects localized evolutionary dynamics driven by environmental factors, host specificity, and vector variability (Maina et al., 2017, 2019; Ahsan et al., 2023). Previous studies reported by Ullah et al. (2003), Weber and Bujarski (2015) highlighted that the cp gene is often subjected to positive selection pressures due to its crucial role in host recognition and immune evasion, making it a hotspot for adaptive evolution. In support of this, Pfosser and Baumann (2002) also observed significant variation in the cp gene across ZYMV populations, reinforcing the idea of its evolutionary importance.

Conversely, isolates from Brazil and North America showed relatively high genetic similarity with ASSM-2025; particularly in the polyprotein and cp genes. Brazilian strains displayed nucleotide identities between 92.47 % and 93.31 %, suggesting limited divergence, potentially due to stable virus-host dynamics or reduced selective pressures in those agro-ecosystems (Romay et al., 2014). These findings affirm that, despite some regional variability, ZYMV retains a conserved genomic core; particularly in functionally essential regions.

High conservation of the polyprotein and cp genes across global isolates indicates strong purifying selection, which helps maintain their structural and functional integrity. This makes them reliable molecular markers for diagnostic development. Identifying conserved domains within these genes facilitates the creation of universal primers and probes, essential for effective and comprehensive ZYMV detection; particularly as the virus spreads and diversifies (Sanfaçon, 2020). The regional variability, especially in Australian isolates, highlights the need for continuous molecular surveillance and localized epidemiological studies to detect emerging variants that may bypass current diagnostic tools or exhibit altered pathogenicity (Jones, 2014; Ladner et al., 2014). Additionally, understanding viral dynamics is crucial for improving integrated pest management (IPM) strategies and guiding breeding programs to enhance host resistance to local strains (Lecoq and Desbiez, 2012; Jones, 2014; Tatineni and Hein, 2023).

The observed genetic conservation of the polyprotein and cp genes highlights their functional importance across global ZYMV strains. These regions are crucial for viral replication, virion assembly, and host interactions, making them ideal candidates for diagnostic assay development (Glasa et al., 2006). However, regional variability, especially in Oceania and Egypt, suggests that new viral strains may evolve to evade current detection methods or present challenges for resistance breeding (Jones, 2014). For instance, the lower sequence identity of Australian and Egyptian isolates in the cp gene indicates the possibility of local evolutionary pressures shaping viral diversity (Alinizi et al., 2021). This calls for strain-inclusive diagnostic tools that consider regional sequence variations to ensure sensitivity and reliability in virus detection.

Moreover, identifying both conserved and divergent regions in the viral genome is essential for guiding resistance breeding strategies. The polyprotein and cp genes; coupled with varying sequence divergence across regions, emphasize the need for region-specific strategies when selecting for resistant cultivars. For example, breeding programs in Oceania may need to address the unique viral pressures in Australia, while in Africa, lower variability in cp genes may allow for more standardized breeding approaches (Lecoq and Desbiez, 2012). These findings highlight the importance of adopting an integrated approach to diagnostic tool development and resistance breeding, taking into account both genetic conservation and regional diversity (Jones, 2014).

Zucchini yellow mosaic virus isolates from various regions exhibited high conservation; particularly in key functional domains like the polyprotein and coat protein. For example, Egyptian isolates; especially Egz1 (MT383104.1), showed 98.71 % nucleotide identity, indicating strong genetic similarity to the reference strain. This conservation suggests that these proteins are under selective pressure to maintain their function, ensuring the effectiveness of existing diagnostic primers across the diverse strains (Glasa and and Pittnerová, 2006). However, regional variations were observed; particularly in African and Asian isolates, which exhibited more amino acid substitutions than European and American isolates (Alinizi et al., 2021). These differences; especially in the coat protein, may reflect local host adaptations and provide insights into ZYMV’s evolutionary dynamics and potential spread (Lecoq and Desbiez, 2012; Jones, 2014). The observed genetic diversity underscores the need for ongoing surveillance to monitor virus spread, identify emerging strains, and track changes that could impact transmissibility or resistance (Fargette et al., 2006). Surveillance targeting region-specific variants can inform the development of resistant cultivars and optimize pest management strategies to mitigate ZYMV’s agricultural impact (Tatineni and Hein, 2023).

This analysis, incorporating the Egyptian ASSM-2025 reference strain and global comparison of ZYMV isolates, offers a significant advancement over previous regional studies. By examining isolates from diverse regions, we provide a comprehensive understanding of the virus’s genetic diversity, evolutionary dynamics, and regional adaptations. This expanded scope allows for identification of conserved and variable regions, improving diagnostic accuracy, and revealing virus’s potential for adaptation (Desbiez and Lecoq, 1997; Farag et al., 2024). This comparison suggests that existing diagnostic primers targeting conserved regions, such as the polyprotein and coat protein, will remain effective across strains (Kumar et al., 2018; Abdel-Razek et al., 2025), supporting the development of universal diagnostic standards. Additionally, these insights inform biosecurity protocols, guide surveillance efforts, and help shape crop management strategies to mitigate ZYMV’s spread and economic impact globally (Lecoq and Desbiez, 2012; Abdel-Razek et al., 2025).

Variability in the cp gene of ZYMV can impact host-virus interactions, immune evasion, and viral fitness. Amino acid substitutions in the cp gene may influence virus binding to host receptors, altering host range and transmission dynamics (Desbiez et al., 2002; Moya-Ruiz et al., 2023). Changes in epitope regions could enable the virus to evade host immune responses, enhancing its persistence (Dunham et al., 2014; Alinizi et al., 2021). Positively selected sites within the cp gene, if being identified, may indicate adaptation to specific hosts or environmental conditions, providing insights into viral evolution (Krause-Sakate et al., 2005; Maghamnia et al., 2018). These variations have diagnostic and epidemiological significance, as they may affect the effectiveness of current diagnostic primers and inform the surveillance efforts done to track emerging strains, aiding in the development of targeted crop management strategies and biosecurity protocols (Abdel-Razek et al., 2025).

The significant variability observed in the ZYMV cp gene poses diagnostic challenges, particularly in regions with high amino acid substitutions, leading to false negatives in traditional RT-PCR or ELISA-based assays (Krishnan et al., 2022). To address this, diagnostic tools should focus on conserved regions, such as the polyprotein, which are less likely to mutate, enhancing detection sensitivity (Khanal et al., 2021). These conserved motifs can also be used in epitope-based assays or next-generation sequencing (NGS) panels, allowing for more precise detection and monitoring of viral evolution (Kondratenko et al., 2021; Carivali et al., 2025). Additionally, these findings can inform resistance breeding strategies by targeting stable cp regions, facilitating the development of virus-resistant cucurbit varieties and identification of candidate resistance genes, offering more durable and broad-spectrum protection (Maghamnia et al., 2018).

This study has several limitations, including reliance on partial sequences, lack of experimental validation for predicted antigenic regions, and potential sampling bias. Future research should include functional studies on divergent cp variants, broader sampling in underrepresented regions like Central Asia and Sub-Saharan Africa, and development of diagnostic kits based on conserved epitopes for better ZYMV detection.

Experimental validation

The bioinformatics findings presented in this study were derived from robust sequence alignment and comparative analysis using internationally recognized databases and tools. While this research is primarily computational, the identified patterns of genetic diversity and conserved protein domains lay on a strong foundation for future experimental validation. Laboratory-based assays, including RT-PCR, protein expression, and host-virus interaction studies are planned to confirm functional relevance of the observed sequence variations; particularly those found in the coat protein region of the divergent isolates.

Conclusions and Recommendations

This global comparative study of Zucchini yellow mosaic virus (ZYMV) isolates represents the first comprehensive analysis integrating both nucleotide and protein-level comparisons across six continents using an Egyptian reference strainS (ZYMV ASSM-2025). It reveals a high degree of conservation in polyprotein and coat protein genes; despite notable nucleotide variability in several regions such as the Oceania and the Americas. These variations; particularly in the coat protein, suggest regional adaptation influenced by host diversity and environmental pressures. The findings of this study underscore its novelty in uncovering region-specific molecular signatures and highlight the need for continuous genomic surveillance and tailored management strategies.

We recommend updating molecular diagnostic tools to reflect emerging variants, integrating regional viral data into resistance breeding programs, and enhancing international collaboration to monitor and control ZYMV spread. Such efforts are vital to protecting cucurbit crops and ensuring global food security. Moreover, this comparative analysis offers valuable insights into ZYMV’s genetic diversity. It is acknowledged that the inclusion of partial sequences may limit the generalizability of certain findings. Future studies incorporating more complete genomes from underrepresented regions are recommended to provide a more comprehensive understanding of ZYMV evolution and strain-specific dynamics.

Acknowledgments

We sincerely acknowledge Dr. Samar S.A. El-Masry for her valuable contribution to bioinformatics analysis and insightful guidance throughout this study.

Novelty Statement

This study represents the first comprehensive comparative analysis of Zucchini yellow mosaic virus (ZYMV) isolates across six global regions using an Egyptian reference strain (ZYMV ASSM-2025) as a benchmark. By integrating both nucleotide and protein-level analyses of polyprotein and coat protein genes, it uncovers region-specific patterns of genetic diversity and highlights unique molecular signatures in North American and Australian isolates. These findings reveal previously unreported regional adaptations and provide essential genomic insights to inform diagnostic assay development, resistance breeding, and targeted disease management strategies for ZYMV worldwide.

Author’s Contribution

SEEF: Conceptualization, data curation, investigation.

SI: Data curation, investigation, validation.

MHA: Conceptualization, data curation, investigation, roles/writing original draft.

AS: Data curation, investigation, roles/writing original draft, writing review and editing.

All authors approved the final manuscript.

Ethical approval

Ethical approval was not required for this study, as it involved only plant and microbial materials that do not fall under human or animal ethics regulations.

Funding source

This research was conducted without any external financial support.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.NRMJ/........

Conflict of interests

The authors have declared no conflicts of interest.

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