Case presentation and laboratory diagnosis
On November 4, 2021, a 9-month-old male infant presented a two-day fever and maculopapular rash, clinically suspected of measles virus infection, was admitted to Dashti Healthcare Center, Bushehr, Iran. He has not received the MMR vaccine, as the first shot of the vaccine administers to children who are 12 months old. There was no information available regarding the past medical or travelling history of the patient. Upon admission, a nasopharyngeal swab, urine and serum specimen were collected which the first two samples, where placed in VTM (viral transport media). These specimens were sent at a low temperature (4℃) to the National Measles and Rubella Center for measles virus serological, cellular and molecular examination according to the CDC and WHO protocols [11] . At first, Anti-Measles virus IgM Elisa test (EUROIMMUN,Germany) was performed on the serum specimen in which the Elisa test was positive. For further investigation, the nasopharyngeal sample was cultured in Vero-hSLAM cell line, to see the specific cytopathic effect (CPE) of measles virus –the syncytia. Then the molecular detection was performed, as the following: total genomic ribonucleotide acid was extracted using High Pure Viral Nucleic acid kit (Roche Diagnostics GMbH, Germany) according to the manufacturer’s instructions. To confirm the serology result, the extracted RNA was tested for measles virus using the 1-step RT-PCR Master mix (Biotechrabbit, Germany) by using specific primers (MeV214 and MeV216). Gel electrophoresis of the PCR product showed an approxiamtely 610 bp band which were assessed by sanger sequencing using Genetic analyzer (3130, Applied Biosystems, USA). Nucleotide analysis confirmed indicated that the measles virus belonged to the B3 strain (based on C-‎terminal of the nucleocapsid protein ), which is currently circulating in Iran. Concurrently, we investigated the nasopharyngeal swab for influenza virus and SARS-CoV-2 to determine whether the patient had a co-infection. For influenza virus and SARS-CoV-2 detection, two separated real-time RT-PCR reactions using specific primers and probes and Invitrogen (USA) master mix were used. The results showed that, the patient was positive for influenza A virus with a cycle threshold of 32, and was negative for SARS-CoV-2. To find out the subtype of this influenza virus strain, another qRT-PCR (Invitrogen,USA) targeting the HA gene was performed, which results in influenza virus A/H3N2 identification.