**1. Introduction**

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single-stranded, positive-sense RNA virus, which belongs to the genus *Porartevirus*, of the family *Arteriviridae* [1,2]. The genome of PRRSV is approximately 15 kb in length and contains over 10 open reading frames (ORFs) [3]. The ORF1a and ORF1b of PRRSV account for two-thirds of its genome and encode all non-structural proteins, which are required for viral replication, while ORFs 2–7 encode structural proteins [3]. Based on sequence similarity, all PRRSV isolates within the genus *Porartevirus* were further classified into two species: *PRRSV-1* and *PRRSV-2* [1,2]. *PRRSV-1* and *PRRSV-2* strains share approximately 60% nucleotide sequence identity and exhibit serotype differences [4,5]. PRRSV infection in pigs is highly restricted to cells of monocyte-macrophage lineages, such as pulmonary alveolar macrophages (PAMs) [6,7]. It is believed that PRRSV infection in susceptible cells

is mediated by several cellular receptors or factors [8], such as heparin sulfate (HS) [9], vimentin [10], Cluster of differentiation (CD)151 [11], porcine CD163 (CD163) [12], sialoadhesin (CD169) [13], Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin(DC-SIGN, also called CD209) [14] and myosin-9 heavy chain(MYH9) [15]. However, only CD163 and MYH9 are indispensable for PRRSV infection [15,16].

Current strategies for PRRS control are inadequate, although substantial efforts have been dedicated to controlling this disease. Since the first report of PRRS in the United States in 1987, PRRS remains one of the major challenges to the global swine industry, while vaccines against PRRS have been commercially available for over two decades [17–19]. This suggests the urgent need for novel methods for PRRSV control and prevention, as well as a deep understanding of viral–host interactions between PRRSV and its permissive cells.

Cationic polymers, which have been used as DNA transfection reagents via facilitating the entry of DNA to target cells [20,21], are also known to have potent antimicrobial activity [22,23]. Polyethylenimine (PEI) is one such cationic polymer, and its novel anti-viral activity has been investigated in several studies [24–27]. Since PEI can be either a linear or branched molecule with a broad molecular weight range, different PEI molecules have great divergence on modulating viral replication. *N*,*N*-dodecyl, methyl-PEI (DMPEI), synthesized from a commercial 750 kDa branched PEI, has been demonstrated to significantly reduce the infectivity of wild-type and drug-resistant influenza A virus [24], as well as HSV-1 and HSV-2 [25]. Meanwhile, the 25 kDa linear PEI is highly effective against human papillomaviruses (HPVs) and human cytomegaloviruses (HCMVs) [26]. However, another report suggests that the 70 kDa branched PEI accelerates HIV-1 infection despite its inhibition of HIV-1 attachment to cells [27]. This paradoxical effect of PEI on HIV infection is not fully understood. The effect of PEI on PRRSV proliferation is not known.

The objective of this study was to explore PEI to inhibit the replication of PRRSV. Our results demonstrate that the 40 kDa linear PEI, but not branched PEI, exhibited a broad-spectrum inhibition of heterogeneous *PRRSV-2* isolates in both MARC-145 cells and primary porcine pulmonary alveolar macrophages (PAMs). Furthermore, we investigated mechanisms of the 40 kDa linear PEI in the inhibition of PRRSV. Our results showed that PEI could reduce the attachment of PRRSV to the susceptible cells. PEI had a minimal effect in the internalization of PRRSV in MARC-145 cells but promoted the internalization of PRRSV into PAMs. This suggests that the internalization process of PRRSV virions into these two types of cells may be different.
