Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia, from 1995 to 2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1 to 3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza virus vaccine strains. While most influenza B virus lineages in Malaysia were short-lived, one circulated for 3 to 4 years.
Reassortment of genetic segments between and within influenza B lineages (Victoria and Yamagata) has been shown to generate novel reassortants with unique genetic characteristics. Based on hemagglutinin (HA) and neuraminidase (NA) genes, recent surveillance study has identified reassortment properties in B/Phuket/3073/2013-like virus, which is currently used in the WHO-recommended influenza vaccine. To understand the potential reassortment patterns for all gene segments, four B/Phuket/3073/2013-like viruses and two unique reassortants (one each from Yamagata and Victoria) detected in Malaysia from 2012-2014 were subjected to whole-genome sequencing. Each gene was phylogenetically classified into lineages, clades and sub-clades. Three B/Phuket/3073/2013-like viruses from Yamagata lineage were found to be intra-clade reassortants, possessing PA and NA genes derived from Stockholm/12-like sub-clade, while the remaining genes from Wisconsin/01-like sub-clade (both sub-clades were within Yamagata Clade 3/Yam-3). However, the other B/Phuket/3073/2013-like virus had NS gene that derived from Stockholm/12-like sub-clade instead of Wisconsin/01-like sub-clade. One inter-clade reassortant had Yamagata Clade 2/Yam-2-derived HA and NP, and its remaining genes were Yam-3-derived. Within Victoria Clade 1/Vic-1 in Victoria lineage, one virus had intra-clade reassortment properties: HA and PB2 from Vic-1B sub-clade, MP and NS from a unique sub-clade "Vic-1C", and the remaining genes from Vic-1A sub-clade. Although random reassortment event may generate unique reassortants, detailed phylogenetic classification of gene segments showed possible genetic linkage between PA and NA genes in B/Phuket/3073/2013-like viruses, which requires further investigation. Understanding on reassortment patterns in influenza B evolution may contribute to future vaccine design.
Epidemiological and evolutionary dynamics of influenza B Victoria and Yamagata lineages remained poorly understood in the tropical Southeast Asia region, despite causing seasonal outbreaks worldwide. From 2012-2014, nasopharyngeal swab samples collected from outpatients experiencing acute upper respiratory tract infection symptoms in Kuala Lumpur, Malaysia, were screened for influenza viruses using a multiplex RT-PCR assay. Among 2,010/3,935 (51.1%) patients infected with at least one respiratory virus, 287 (14.3%) and 183 (9.1%) samples were tested positive for influenza A and B viruses, respectively. Influenza-positive cases correlate significantly with meteorological factors-total amount of rainfall, relative humidity, number of rain days, ground temperature and particulate matter (PM10). Phylogenetic reconstruction of haemagglutinin (HA) gene from 168 influenza B viruses grouped them into Yamagata Clade 3 (65, 38.7%), Yamagata Clade 2 (48, 28.6%) and Victoria Clade 1 (55, 32.7%). With neuraminidase (NA) phylogeny, 30 intra-clade (29 within Yamagata Clade 3, 1 within Victoria Clade 1) and 1 inter-clade (Yamagata Clade 2-HA/Yamagata Clade 3-NA) reassortants were identified. Study of virus temporal dynamics revealed a lineage shift from Victoria to Yamagata (2012-2013), and a clade shift from Yamagata Clade 2 to Clade 3 (2013-2014). Yamagata Clade 3 predominating in 2014 consisted of intra-clade reassortants that were closely related to a recent WHO vaccine candidate strain (B/Phuket/3073/2013), with the reassortment event occurred approximately 2 years ago based on Bayesian molecular clock estimation. Malaysian Victoria Clade 1 viruses carried H274Y substitution in the active site of neuraminidase, which confers resistance to oseltamivir. Statistical analyses on clinical and demographic data showed Yamagata-infected patients were older and more likely to experience headache while Victoria-infected patients were more likely to experience nasal congestion and sore throat. This study describes the evolution of influenza B viruses in Malaysia and highlights the importance of continuous surveillance for better vaccination policy in this region.
Micro RNAs (miRNAs) are a class of small non-coding single-stranded RNA, which play an important role in modulating host-Influenza A virus (IAV) crosstalk. The interplay between influenza and miRNA interaction is defined by a plethora of complex mechanisms, which are not fully understood yet. Here, we demonstrate that in IAV infected A549 cells, a synchronous increase was observed in the expression of mTOR up to 24 hpi and significant downregulation at 48 hpi. Additionally, NP of IAV interacts with mTOR and modulates the levels of mTOR mRNA and protein, thus regulating the translation of host cell. RNA sequencing and qPCR analysis of IAV-infected A549 cells and NP transfected cells revealed that miR-101 downregulates mTOR transcripts at later stages of infection. Ectopic expression of miR-101 mimic led to a decrease in expression of NP, a reduction in IAV titer and replication. Moreover, treatment of the cells with Everolimus, a potent inhibitor of mTOR, resulted in an increase of miR-101 transcript levels, which further suppressed the viral protein synthesis. Collectively, the data suggest a novel mechanism that IAV stimulates mTOR pathway at early stages of infection; however, at a later time-point, positive regulation of miR-101 restrains the mTOR expression, and hence, the viral propagation.
Bats are special in their ability to host emerging viruses. As the only flying mammal, bats endure high metabolic rates yet exhibit elongated lifespans. It is currently unclear whether these unique features are interlinked. The important inflammasome sensor, NLR family pyrin domain containing 3 (NLRP3), has been linked to both viral-induced and age-related inflammation. Here, we report significantly dampened activation of the NLRP3 inflammasome in bat primary immune cells compared to human or mouse counterparts. Lower induction of apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and secretion of interleukin-1β in response to both 'sterile' stimuli and infection with multiple zoonotic viruses including influenza A virus (-single-stranded (ss) RNA), Melaka virus (PRV3M, double-stranded RNA) and Middle East respiratory syndrome coronavirus (+ssRNA) was observed. Importantly, this reduction of inflammation had no impact on the overall viral loads. We identified dampened transcriptional priming, a novel splice variant and an altered leucine-rich repeat domain of bat NLRP3 as the cause. Our results elucidate an important mechanism through which bats dampen inflammation with implications for longevity and unique viral reservoir status.
We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.