Human parainfluenza virus type 4b (HPIV-4b) is an under-recognized but clinically significant pathogen associated with acute respiratory tract infections (ARTIs), particularly in young children. Its molecular and epidemiological profiles remain poorly characterized in resource-limited Pakistani settings. This study aimed to investigate the prevalence, clinical associations, and molecular characteristics of HPIV-4b among children under five years of age presenting with respiratory illnesses in the Cholistan region of Pakistan.

Between October 2022 and March 2025, 200 nasopharyngeal swabs were collected from pediatric patients exhibiting respiratory symptoms. Samples were screened for HPIV-4b using multiplex reverse transcriptase PCR (mRT-PCR). Positive samples were subjected to further molecular analysis of the hemagglutinin-neuraminidase (HN) and fusion (F) genes. Selected strains underwent sequencing and phylogenetic analysis.

HPIV-4b was detected in 80 (40 %) of the children, with the highest positivity observed in infants aged 0–1 year. A higher infection rate was noted in male children (62.5 %). Clinical features associated with infection included fever, barking cough, and wheezing (p < 0.05). The virus was more prevalent among children from socioeconomically disadvantaged families and those with a family history of pulmonary disorders. Sequence data revealed high similarity between local strains and international isolates from Japan, the USA, and China. Phylogenetic analysis indicated the circulation of genetically distinct but evolutionarily conserved HPIV-4b lineages.

This is the first molecular and epidemiological characterization of HPIV-4b in Pakistani children, highlighting its clinical relevance and genetic diversity. The findings underscore a need for early diagnosis and sustained genomic monitoring.