Geta virus (GETV) is an emerging mosquito-borne virus that threatens a wide range of animals and humans. From July to September 2024, a concentrated outbreak of GETV occurred in pig farms across Henan Province, marking one of the most widespread and concentrated outbreaks in mainland China in recent years.
From July to September 2024, a concentrated outbreak of Geta virus (GETV) occurred in multiple pig farms in Henan Province. Twenty-seven strains were isolated from 31 pig farms, 22 of which formed a unique cluster in the phylogenetic tree and exhibited amino acid mutations in the nsP3 and E2 proteins.
Experiments demonstrated that this GIII variant exhibited significantly enhanced pathogenicity in piglets, with a 100% lethality rate, and also exhibited high pathogenicity in mice. This study is the first systematic investigation of a large-scale GETV outbreak in Henan pig farms, revealing the enhanced virulence and molecular characteristics of the GIII variant.
Introduction
Geta virus (GETV) is a single-stranded, positive-sense RNA virus that infects a wide range of animals and may even pose a threat to humans. From July to September 2024, a concentrated outbreak of GETV occurred in commercial pig farms in Henan Province, resulting in the isolation of 27 strains. The GIII variant was the predominant pathogenic strain, exhibiting increased virulence compared to earlier strains. This study provides a reference for understanding the molecular epidemiology and pathogenicity of GETV.
Materials and Methods
This study used BHK-21 and PK-15 cells to culture the porcine GETV isolate HNJZ-S1. Viral nucleic acid was extracted from suspected infected pig farms in Henan and Shanxi, and cDNA was synthesized. Viral detection was performed by PCR/RT-PCR. Positive samples were isolated, purified, and identified in cells. Viral growth characteristics and plaque formation were determined. The E2 gene and the entire genome were amplified, cloned, and sequenced for sequence alignment and phylogenetic analysis.
In animal experiments, GETV-negative piglets and two-day-old SPF suckling mice were injected intramuscularly or subcutaneously with the virus. Clinical signs or mortality were observed, and viral loads in blood and tissues were measured. Viral RNA was extracted from tissue samples, cDNA was synthesized, and then detected by SYBR Green qPCR. Data are presented as mean ± SD and analyzed using GraphPad Prism 9.0.
Research Results
1. GETV Sample Collection and Virus Detection
In July 2024, a high-mortality outbreak occurred in 3- to 10-day-old piglets at a pig farm in Zhumadian, Henan Province, with a morbidity rate of 30% and a mortality rate of 80%. Laboratory confirmation indicated that the outbreak was caused by a single GETV strain. Subsequently, suspected GETV outbreaks occurred at pig farms across Henan Province. Clinical sample collection revealed outbreaks in 12 of 18 prefecture-level cities and 21 of 157 counties (Figure 1(a)). From July to September, there were one, 15, and 16 outbreaks, respectively. The main symptoms in piglets were severe diarrhea, ataxia, and hind limb weakness. Autopsies revealed swollen and hemorrhagic lymphadenopathy, pulmonary hemorrhages, scattered superficial hemorrhages, and subcutaneous edema (Figure 1(b)). Of the 36 GETV-positive samples, only two were co-infected with PDCoV, PCV2, or JEV; the remainder were infected with a single GETV (Figure 1(c)).
Figure 1. Overview of the GETV Outbreak in Pig Farms in Henan Province
(a) Distribution of GETV-positive pig farms in Henan Province. (b) Gross histological lesions in infected piglets during this outbreak.
(c) PCR identification of GETV and other viruses in tissue samples.
Conclusion: GETV infection occurred in piglets at multiple farms in Henan Province, primarily caused by a single virus. Clinical manifestations included diarrhea, ataxia, and hind limb weakness, with mixed infections occurring in a few samples.
2. Isolation, Identification, and Titer Determination of GETV
From 36 positive samples, 28 GETV strains were successfully isolated in BHK-21 cells. HNzk-XH1 and HNzmd-XP1 were identified as GETV. After 48 hours of infection in BHK-21 or PK-15 cells, significant cytopathic changes, including cell shrinkage, rounding, and exfoliation, were observed compared to controls. RT-PCR and electron microscopy further confirmed the viral identity. The proliferation curves showed that the growth kinetics of the two virus strains were similar, with titers reaching a peak at 36 hours after infection and then declining. Plaque assay results showed that the plaques of the two virus strains were similar in size, but differed in number.
Figure 2. Isolation and Characterization of GETV
(A) After infection of BHK-21 and PK-15 cells with HNzk-XH1 or HNzmd-XP1, cytopathic effects were observed within 12 to 36 hours.
(B) GETV isolates were identified by RT-PCR. (C) GETV particle morphology was examined by electron microscopy.
(d) Growth of HNzk-XH1 or HNzmd-XP1 in BHK-21 and PK-15 cells. (e) Plaque morphology of GETV on BHK-21 cells.
Conclusion: GETV strains HNzk-XH1 and HNzmd-XP1 were successfully isolated from swine farm samples. Both strains caused significant cytopathic effects in cells, with similar growth kinetics and titer variations, but with differences in plaque numbers.
3. Sequencing and Phylogenetic Analysis
The whole-genome sequences (GenBank accession numbers: PQ658739–PQ658750) and E2 gene sequences (GenBank accession numbers: PQ658751–PQ658766) obtained in this study have been submitted to GenBank. Whole-genome homology analysis showed that the nucleotide identity among the isolates ranged from 98.4% to 100.0%, and the amino acid identity ranged from 99.3% to 100.0%. Compared with the reference strain, the nucleotide identities of these isolates with GI, GII, GIV, and GIII were 94.8%–95.0%, 96.9%–97.0%, 95.6%–97.3%, and 96.9%–99.8%, respectively. The amino acid identities were 98.5%–98.8%, 98.9%–99.1%, 98.3%–99.5%, and 98.7%–100.0%, respectively.
Phylogenetic analysis based on the E2 genes of 28 isolates and 12 whole genomes revealed that all isolates belonged to GIII, distantly related to other groups (Figure 3(a,b)). Most of them clustered with the GDHYLC23 variant (Figure 3(b)). Amino acid sequence alignment revealed four unique mutations in the nsP3 and E2 proteins of these isolates (Figure 3(c)). The nsP3 P329S mutation is located in the ZBD region, the A381T and V503G mutations are located in the HVD region, and the E2 protein has a D323E mutation at position 323. Based on these characteristics, the isolates in this clade were identified as GIII variants.
Figure 3. Phylogenetic analysis and amino acid sequence alignment of isolates. Phylogenetic analysis of isolates based on E2 (a) and complete genome (b).
Conclusion: The isolates in this study all belong to the GIII genotype, distantly related to other genotypes, and possess unique amino acid mutations in the nsP3 and E2 proteins, confirming that these isolates are GIII variants.
4. Pathogenicity of GETV HNzk-XH1 Strain in Piglets
Due to the high mortality rate of GETV outbreaks in commercial pig farms and the high titers of GETV viruses isolated from BHK-21 and PK-15 cells, the higher-titer HNzk-XH1 strain was selected for pathogenicity evaluation in this study. Piglets in the challenge group were intramuscularly injected with 10⁷TCID₅₀ of the HNzk-XH1 strain, while the control group was injected with an equal volume of DMEM medium. Twenty-four hours after challenge, piglets in the challenge group developed diarrhea, followed by hind limb paralysis 1.5 days later. Two days later, some piglets died, and within four days, all piglets had died, reaching a 100% mortality rate (Figure 4(c)). Clinical symptom scores showed that the HNzk-XH1 group had significantly more severe symptoms than the control group (Figure 4(b)). Dead piglets were autopsied immediately, while the control group was autopsied 8 days later. The small intestine wall of the HNzk-XH1 group was significantly thinned, with yellow, watery contents, while the small intestine of the control group was normal (Figure 4(a)). RT-qPCR analysis revealed that the GETV nsP1 gene copy number in blood collected two days after challenge was close to 10⁶/ml, while no virus was detected in the control group (Figure 4(d)). Examination of the liver, lung, kidney, spleen, small intestine, and brain tissues of the piglets revealed high viral loads in all organs of the HNzk-XH1-infected group, while no GETV RNA was detected in the control group (Figure 4(e)).
Figure 4. Pathogenicity in Piglets
(a) Clinical signs and autopsy results of infected piglets. (b) Clinical scores of infected piglets.
(c) Survival rate of infected piglets. (d) Viral RNA levels in whole blood of infected piglets on day 2 post-infection.
(e) Viral RNA levels in different organs of piglets that died or were euthanized after infection.
Conclusion: The HNzk-XH1 strain is highly pathogenic to piglets, rapidly causing severe clinical symptoms and mortality. The virus is widely present in the blood and major organs. No abnormalities were observed in the control group.
5. Pathological changes in suckling mice infected with the HNzk-XH1 variant
This study demonstrates that suckling mice are an ideal model for studying the virulence of GETV. The experimental results showed that infection of suckling mice with the HNzk-XH1 variant caused hind limb paralysis and growth retardation (Figure 5(a)), and led to rapid death at different doses. In contrast, the HNZJ-S1 strain was less lethal, and suckling mice infected with low doses survived and gained normal weight, indicating that there was a significant difference in the pathogenicity of the two strains in suckling mice.
Figure 5. Comparison of Pathogenicity of HNzk-XH1 and HNZJ-S1
(a) Clinical symptoms of infection in suckling mice. (b) Survival rate of suckling mice infected with different doses of the HNzk-XH1 strain.
(c) Survival rate of suckling mice infected with different doses of the HNZJ-S1 strain. (d) Weight changes of suckling mice infected with different doses of the HNzk-XH1 strain.
(e) Weight changes of suckling mice infected with different doses of the HNZJ-S1 strain.
Conclusion: The HNzk-XH1 variant is highly pathogenic in suckling mice, causing hind limb paralysis, growth retardation, and rapid death. The HNZJ-S1 strain is less pathogenic, and suckling mice infected with low doses survive and maintain normal body weight, indicating a significant difference in virulence between the two strains.
Conclusion
This study demonstrates that the GETV outbreak in pig farms in Henan Province from July to September 2024 was identified as an independent, branching variant of the GIII group, harboring four unique amino acid mutations in the E2 and nsP3 proteins. Compared to earlier isolates, this variant exhibits enhanced virulence and may have been quietly transmitted through pigs and mosquitoes prior to the outbreak. Climate conditions and pig farm circulation patterns accelerated the spread of the epidemic. Currently, there is no vaccine available in China, necessitating enhanced surveillance and timely adjustments to prevention and control strategies.
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