Rabies is currently regarded as incurable, with a nearly 100% mortality rate once clinical symptoms manifest. Timely and standardized postexposure prophylaxis (PEP) can effectively prevent rabies onset [3]. However, many rabies victims are not able to obtain PEP in time[4]. A study assessing 10,971 rabies cases in China between 2006 and 2012 reported that only 35.0% of patients treated the wound, only 11.7% received PEP vaccination, and only 3.0% were injected with rabies immunoglobulin [5]. In 2005, a retrospective survey of 885 rabies patients in 5 provinces of China revealed that 60.6% of the patients failed to receive wound treatment, 49.0% of the patients did not receive PEP vaccination, and 96.16% did not receive immunoglobulin treatment [6].
In this study, Patients 1 and 3 experienced Level III exposure; however, Patient 1 did not receive wound care, vaccination, or passive immunization after exposure. Similarly, Patient 3 only undertook self-care for the wound and did not receive vaccination or passive immunization. Patients 2, 4, and 5 lacked a clear history of rabies exposure. Notably, Patients 4 and 5 included cats and dogs but denied any bites or scratches from these animals and reported that none of their pets had been vaccinated against rabies. Overall, adherence to PEP among these patients was alarmingly low and inconsistent. This suggests the need for further publicity and education to raise awareness of rabies among the population and to improve the accessibility and availability of PEP treatment for rabies.
In China, comprehensive guidelines for rabies PEP have been established. However, practical implementation is hindered by the high cost of rabies immunization products, leading some individuals with dog bites to forgo treatment, particularly those from economically disadvantaged backgrounds. Some economically advanced provinces and cities in China have progressively included rabies PEP in medical insurance coverage, significantly increasing the effectiveness of rabies prevention and control measures. On the basis of both domestic and international experiences in rabies control, it is recommended that government authorities incorporate rabies PEP into medical insurance coverage, where feasible, to reduce the financial burden on individuals with dog bites and further support the achievement of rabies control objectives[7].
In the absence of a relevant exposure history or typical symptoms, the clinical diagnosis of rabies is often unreliable. Thus, laboratory testing is essential for confirming the diagnosis [3]. The World Health Organization (WHO) recommended the direct fluorescent antibody (DFA) test as the “gold standard” for rabies diagnosis [8]. However, DFA requires stringent standards for laboratory equipment and personnel. Currently, methods such as RT‒PCR and real-time PCR are more widely used, yet they are susceptible to false negatives and false positives, complicating the rapid laboratory confirmation of suspected or clinically diagnosed cases. In this study, CSF and serum samples from Cases 2, 4, and 5, which were collected at one-day intervals, tested positive with mNGS but negative with real-time PCR. These findings suggest that mNGS can facilitate early and rapid diagnosis of rabies, demonstrating robust clinical utility and warranting further adoption. However, the limited data available necessitate further research to evaluate its specificity and sensitivity.
The WHO expert consultation on rabies recommends that laboratory diagnostic samples for rabies include secretions, biological fluids (such as saliva, CSF, and serum), and specific tissues (such as skin biopsy samples, including hair follicles from the nape of the neck). Although brain tissue remains the preferred postmortem diagnostic specimen [3], obtaining such samples, as well as skin biopsy samples, can be challenging in clinical settings. Consequently, detecting viral RNA is a critical method for diagnosing rabies, with saliva samples being particularly favored for viral nucleic acid testing[9]. In this study, real-time PCR technology was used to analyze 15 samples (8 saliva, 2 CSF, and 5 serum samples) from Cases 1–5. Saliva samples presented the highest positive detection rate (87.5%), whereas CSF and serum samples tested negative. Saliva samples were positive as early as 2 days and as late as 16 days postonset. These results are consistent with those reported by Li H et al. [10]. Despite their convenience and high positivity rate in clinical settings, saliva samples exhibit intermittent viral shedding. Therefore, it is recommended that patients provide three or more consecutive saliva samples for testing. Furthermore, mNGS analysis detected RABV in CSF collected between 2 and 12 days postonset (1–5 days before death) and in serum collected 11 days postonset (4 days before death). Therefore, collecting and analyzing multiple samples from different stages of the disease (e.g., skin, saliva, and urine) can improve detection rates and facilitate earlier diagnosis[11].
Currently, RABV in China is categorized into seven groups, designated China I-VII, on the basis of their epidemiological characteristics, which include differences in prevalence and geographical distribution [12]. Among these groups, the China I group is the most prevalent and widely distributed, representing the dominant strain[13]. In this study, the N gene sequences from four rabies cases were identified as belonging to the predominant China I strain. The nucleotide (amino acid) homology among these four sequences ranged from 99.26%-99.85% (100%). The homology of the encoded proteins was greater than that of the genes, indicating that the mutations observed were primarily synonymous. Furthermore, the composition of the coding genes was consistent with that of other RABVs, with no insertions or deletions detected. These findings suggest that the RABV population circulating in Henan Province in 2024 is relatively homogeneous and has high genetic stability.
The RABV N protein contains four antigenic sites. Except for antigenic site II, the remaining sites are localized to specific regions: antigenic site I spans amino acid residues 358–367; antigenic site III spans residues 313–337; and antigenic site IV spans residues 359–383[14]. In the sequences analyzed in this study, antigenic sites I, III, and IV were highly conserved. The N protein serves as an effective protective antigen capable of inducing antibody responses in B lymphocytes and stimulating T helper lymphocytes. The primary epitopes for T helper cell responses are located at residues 394–408, 404–418, and 21–35, whereas the B-cell epitope is located at residues 369–383[15]. The sequences from the four cases in this study exhibited a high degree of conservation for both T helper cell and B-cell epitopes. Additionally, phosphorylation sites associated with viral RNA transcription and replication, specifically Ser389 and the secondary phosphorylation site Thr375[16–18], were also highly conserved in the analyzed sequences.
In China, the primary fixed RABV strains used for vaccine production are CTN-1, PV, and aG. CTN-1 was isolated from a rabies patient in Zibo, Shandong Province, in 1956. PV was obtained from the Pasteur Institute in France, whereas aG was isolated from a rabid dog in Beiping in 1931[19]. Nucleotide homology analysis of sequences obtained in this study with publicly available vaccine strains revealed values ranging from 85.66–89.87%, with the highest similarity of 89.87% observed with CTN-1. CTN-1 also shows significant homology with domestic street isolates and has a close phylogenetic relationship, confirming its efficacy in preventing rabies in China. Consequently, CTN-1 remains a promising candidate for ongoing and future rabies vaccine production.
Understanding the genetic variations, potential origins, and transmission patterns of RABV is crucial for developing more effective prevention and control strategies. With the continuous emergence of new RABV genotypes, the epidemiology of rabies remains highly complex and distinctive. Therefore, comprehensive molecular epidemiological studies on a larger scale are imperative. These studies should encompass analyzing relationships between RABV isolates and vaccine strains, exploring antigenic differences, monitoring viral strain mutations, and investigating cross-species and interregional transmission patterns of RABV. Furthermore, to achieve the global goal of eliminating human rabies by 2030, widespread public education, enhanced rabies prevention and control measures, the establishment of decentralized animal control networks, and reinforced laboratory-based monitoring are essential. These strategies should incorporate insights gained from rabies elimination efforts in other countries.